bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of formation of complex oligosaccharides by the glucosidase inhibitor bromoconduritol.
Authors: Datema R , Romero P A , Legler G , Schwarz R T
Published: May 2006

The alpha-glucosidase inhibitor bromoconduritol (6-bromo-3,4,5-trihydroxycyclohex-1-ene) inhibits trimming of the innermost glucose residue from the Glc3Man9GlcNAc2 precursor of high-mannose and complex oligosaccharides. This inhibition occurs both in intact cells and with a microsomal enzyme preparation. The formation of lipid-linked oligosaccharides was increased in glucosidase-inhibited cells. Inhibition of transfer of high-mannose oligosaccharides to protein was not observed. In bromoconduritol-treated virus-infected cells, trimming of mannose can occur despite incomplete removal of glucose. The glucosylated high-mannose oligosaccharides GlcMan9GlcNAc, GlcMan8GlcNAc, and GlcMan7GlcNAc were released from viral glycoproteins after digestion with Pronase and endo-beta-N-acetylglucosaminidase H. The formation of complex oligosaccharides was concomitantly inhibited. The release of infectious fowl plague virus particles (an influenza virus) was inhibited from bromoconduritol-treated infected chicken-embryo cells.
Hypolipidemic drugs are inhibitors of phosphatidylcholine synthesis.
Authors: Parthasarathy S , Kritchevsky D , Baumann W J
Published: May 2006

Clofibric acid (CPIB) and several other systemic hypolipidemic drugs are shown to block phosphatidylcholine synthesis by inhibiting cholinephosphotransferase (ChoPTase; CDPcholine:1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2) and particularly lysolecithin acyltransferase (LLAcylTase; acyl-CoA:1-acylglycero-3-phosphocholine O-acyltransferase, EC 2.3.1.23) of rat liver microsomes. Whereas millimolar drug concentrations are required to affect de novo lecithin synthesis catalyzed by ChoPTase, reacylation of lysolecithin by LLAcylTase is inhibited at micromolar levels. Increasing effectiveness in ChoPTase inhibition is observed in the series CPIB, SaH-42-348, tibric acid, S-321328, WY-14643, S-8527, and DH-990, with IC50 ranging from 22 mM (CPIB) to 0.3 mM (DH-990). LLAcylTase inhibition by the hypolipidemic drugs follows the same general pattern, but IC50 concentrations range from 9 mM (CPIB) to 40 microM (DH-990). The agents inhibit ChoPTase (Ki, 25-0.25 mM) and LLAcylTase (Ki, 10-0.025 mM) noncompetitively. The data suggest that inhibition of phosphatidylcholine synthesis, particularly by the LLAcylTase pathway, may be related to a drug's effectiveness in decreasing serum triglyceride and cholesterol levels by blocking lipoprotein synthesis.
Preparation, characterization, and properties of monoclonal antibodies against the lac carrier protein from Escherichia coli.
Authors: Carrasco N , Tahara S M , Patel L , Goldkorn T , Kaback H R
Published: May 2006

Monoclonal antibodies directed against the lac carrier protein purified from the membrane of Escherichia coli were prepared by somatic cell fusion of mouse myeloma cells with splenocytes from an immunized mouse. Several clones produce antibodies that react with the purified protein as demonstrated by solid-phase radioimmunoassay and by immunoblotting experiments; culture supernatants from the clones inhibit active transport of lactose in isolated membrane vesicles. Five stable clones were selected for expansion, formal cloning, and production of ascites fluid, and the antibodies secreted in vivo by each clone also were found to inhibit lactose transport. Antibody from hybridoma 4B1, an IgG2a immunoglobulin, inhibits active transport of lactose in proteoliposomes reconstituted with purified lac carrier and in right-side-out membrane vesicles. In contrast, the antibody has no effect on the generation of the proton electrochemical gradient by membrane vesicles nor does it alter the ability of vesicles containing the lac carrier to bind p-nitrophenyl-alpha-D-galactopyranoside. In order to achieve 50% inhibition of transport activity, a 2- to 3-fold molar excess of antibody to lac carrier is required, regardless of the amount of lac carrier in the membrane. Thus, the concentration of antibody required for a given degree of inhibition is proportional to the amount of lac carrier in the membrane. Finally, antibody-induced inhibition occurs within seconds, an observation suggesting that the epitope is accessible on the surface of the membrane.
Granules 25-30 nm in diameter: basic constituent of the nuclear matrix, chromosome scaffold, and nuclear envelope.
Authors: Engelhardt P , Plagens U , Zbarsky I B , Filatova L S
Published: May 2006

Rat liver nuclear matrix and similar structures derived from isolated Chironomus polytene chromosomes, nuclear envelopes, and intranuclear bodies of frog late oocytes (the karyospheres) were studied by electron microscopy with platinum shadowing and negative staining. We have shown that the treatment of whole nuclei, nuclear envelopes, polytene chromosomes, or karyospheres with nonionic detergent, high salt, and RNase and DNase followed by dilute alkali or hyaluronidase digestion reveals numerous rather uniform granules 25-30 nm in diameter. With omission of the nucleases the granules appear to be associated with DNA strands mostly organized in loops. Many granules form clusters and are arranged in linear or arch-like aggregates or cycles resembling the pore complexes. We suppose that these spherical bodies constitute a basic component of the nuclear matrix, chromosome scaffold, and nuclear envelope and are bound together by hyaluronic acid or some similar glycosaminoglycan.
Specific cleavage of the p15A primer precursor by ribonuclease H at the origin of DNA replication.
Authors: Selzer G , Tomizawa J I
Published: May 2006

We report studies on the mechanism of initiation of DNA replication by p15A, a small plasmid whose origin of replication is known to function much as does that of ColE1. Previous work has shown that an RNA primer for DNA synthesis is generated by the action of RNase H (EC 3.1.26.4) on a precursor transcript. The precursor initiates well upstream of the origin of replication and somehow forms a hybrid with its template during transcription. Here we show that when RNase H cleaves the hybrid at 0 degrees C, an additional cleavage product besides the primer can be identified. Using two-dimensional RNA sequencing techniques, we have established the sequence of this product to within a few nucleotides of each end. The position of the 5' end indicates that the nuclease introduces a nick or very small gap in the precursor at the origin. This suggests that some sequence or structure directs the enzyme to the origin. The position of its 3' end indicates that the precursor terminates at or near a series of six dAs in the template strand about 190 nucleotides from the origin of replication. The data indicate that hybrid formation may be necessary for termination of the precursor at this downstream site.
Isolation of cDNA clones coding for rat tyrosine aminotransferase.
Authors: Scherer G , Schmid W , Strange C M , Röwekamp W , Schütz G
Published: May 2006

Tyrosine aminotransferase (TyrATase; L-tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) from rat liver is subject to glucocorticoid and cAMP as well as developmental control. To isolate DNA sequences encoding TyrATase, we constructed a cDNA library from rat liver poly(A)+RNA enriched for TyrATase mRNA. Recombinant plasmids were screened by differential colony hybridization to poly(A)+RNA isolated from adrenalectomized and dexamethasone-treated animals. Differentially hybridizing plasmids were then shown to contain TyrATase cDNA sequences by their ability to select a mRNA whose in vitro translation product is immunoprecipitable with antiserum against TyrATase. In confirmation, we detect mRNA homologous to TyrATase cDNA sequences in hepatoma cell lines known to contain TyrATase activity but not in a cell line lacking this activity. We show that treatment of rats with dexamethasone or N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate leads to a 5- to 10-fold increase in the amount of TyrATase mRNA.
Correlation of parvalbumin concentration with relaxation speed in mammalian muscles.
Authors: Heizmann C W , Berchtold M W , Rowlerson A M
Published: May 2006

The physiological role of the Ca2+-binding protein parvalbumin in skeletal muscle has been investigated by measuring the parvalbumin content by HPLC in a variety of mammalian muscles, including man, and comparing the results with the respective muscle relaxation properties and fiber type compositions. The parvalbumin concentrations were highest in the skeletal muscles of the smallest animal investigated (mouse, gastrocnemius: 4.9 g/kg), which has the highest relaxation speed, and lowest in the larger animals (horse, deep gluteal muscle: less than or equal to 0.001 g/kg) and man (vastus, triceps: less than or equal to 0.001 g/kg), which have much lower relaxation speeds. Analysis of three type-homogeneous muscles of the guinea pig revealed highest parvalbumin concentrations (0.25 g/kg) in sartorius (type IIB) and lowest concentrations (less than or equal to 0.007 g/kg) in soleus (type I), consistent with the different half-relaxation times of fast and slow muscles. Denervation of the rat extensor digitorum longus, which increases the half-relaxation time from 9.4 to 19 msec, resulted in a 20% decrease of the parvalbumin content. Given this close correlation between parvalbumin content and relaxation speed in a variety of muscles and species, we suggest that parvalbumin is involved directly in the relaxation process in fast muscles.
Role of endogenous cat retrovirus in cell differentiation.
Authors: Rasheed S
Published: May 2006

Several long-term cultures were established from a spontaneous melanoma of a cat. Cells were rounded or spindle shaped and exhibited black/brown pigmentation in the cytoplasm. No virus was released from these cells spontaneously or after treatment with chemicals. However, exogenous infection of the cat melanoma cells with the endogenous cat virus RD114 resulted in remarkable morphological and functional changes. Most of the RD114 virus-infected cells exhibited multiple neuritic extensions and about 1-2% of the population showed characteristics of neuronal cells. Because human, mouse, and hamster melanoma cultures infected with various mammalian retroviruses, including the RD114 virus, did not display any morphological alteration, it is concluded that the neuronal cell differentiation in the cat melanoma cells is a consequence of its specific interaction with the endogenous cat retrovirus.
Interactions of aspirin and other potential etiologic factors in an animal model of Reye syndrome.
Authors: Deshmukh D R , Maassab H F , Mason M
Published: May 2006

Recent studies of Reye syndrome (RS) patients have suggested aspirin treatment as a possible factor in the etiology of this often fatal childhood disorder. the relationship of aspirin treatment to other factors that have been strongly implicated (influenza, ammonia toxicity) cannot be examined directly in patients because aspirin treatment is usually initiated by family members in the prodromal period before RS is diagnosed. In this report we describe the use of an animal model for RS in examining the interactions of these several potential etiological factors. Hyperammonemia and coma were produced in young male ferrets by a brief feeding of an arginine-deficient diet. The effects of influenza infection or aspirin treatment (or both) of control and hyperammonemic ferrets on their serum levels of ammonia, glutamic-oxaloacetic transaminase (GOT;L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), ornithine carbamoyltransferase (OCT; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3), bilirubin, and salicylate were studied. Liver levels of lipids, proteins, and several urea-cycle enzymes were also determined in the comatose ferrets and compared with those of untreated controls and of controls treated with influenza or aspirin, or both. Synergism of these three factors (hyperammonemia, influenza infection, and aspirin treatment) in causing RS-like alterations in these parameters was observed.
Cytoplasmic male sterility in barley: Evidence for the involvement of cytokinins in fertility restoration
Authors: Ahokas Hannu
Published: May 2006

The hypothesis of the association between an increase in cytokinin activity and restoration of anther fertility in
msm1
cytoplasm was tested. The following barley lines with
Hordeum vulgare
cv. Adorra nuclear gene background were studied: Adorra cytoplasm without nuclear restorer gene (fertile), Adorra cytoplasm homozygous for nuclear
Rfm1a
gene (fertile),
msm1
cytoplasm without restorer gene (male sterile),
msm1
cytoplasm homozygous for nuclear
Rfm1a
gene (fertile). Ethanolic extracts of root exudate were fractionated and bioassayed for cytokinins. Both the biological activity and the total quantity of cytokinins appeared lowest in the unrestored male sterile line. The total biological activities of cytokinins in the three fertile lines were similar, but the quantities in the restorer gene carriers appeared lower. On the other hand, the restorer gene carriers, independent of the cytoplasm, showed 8-9 times more of a bound cytokinin. Because the bound form is evidently underestimated by the bioassay, the increase in the bound cytokinin fraction may mean even a higher total content in the
Rfm1a
gene carriers than in Adorra without the gene. The bound cytokinin may be translocated more readily to distal organs (e.g., the anthers) compared with unbound cytokinins. Because cytokinins are associated with various ecophysiological processes, the rise in a particular form may explain the heterogeneous distribution of the restorer gene in wild barley populations in different regions of Israel.
Bent helical structure in kinetoplast DNA
Authors: Marini Joan C. , Levene Stephen D. , Crothers Donald M. , Englund Paul T.
Published: May 2006

We have investigated the unusual physical properties of a restriction fragment of
Leishmania tarentolae
kinetoplast DNA. A gel-purified fragment comprising slightly more than half of a minicircle was determined by Maxam-Gilbert sequence determination to be 490 base pairs (bp) in length. This fragment has dramatically anomalous electrophoretic behavior; it has an apparent size of 450 bp on a 1% agarose gel but migrates as 1,380 bp on a 12% polyacrylamide gel. However, in gel filtration on Sephacryl S-500, the fragment elutes with an apparent size of 375 bp. Finally, it behaves anomalously in electric dichroism experiments. Field-free rotational relaxation times from transient electric dichroism studies are highly sensitive to effective molecular dimensions. The rotational relaxation time of the kinetoplast fragment is smaller than that of a 309-bp control fragment from pBR322. Because rigorous control experiments rule out the possibility that this fragment is modified, these anomalous properties must be dictated by the sequence itself. Fragment behavior indicates that it has an unusually compact configuration; we propose that this molecule contains a region of systematically bent B-DNA. This model accounts for the fragment's difficulty in snaking through the pores of a polyacrylamide gel, its ease in diffusing into Sephacryl beads, and its smaller rotational relaxation time. Bending of this molecule may be caused by periodicities in the DNA sequence.
Molecular cloning of 3-hydroxy-3-methylglutaryl coenzyme a reductase and evidence for regulation of its mRNA.
Authors: Chin D J , Luskey K L , Faust J R , MacDonald R J , Brown M S , Goldstein J L
Published: May 2006

A recombinant plasmid containing a 1.2-kilobase cDNA for 3-hydroxy-3-methylglutaryl coenzyme A reductase was isolated from a cDNA library prepared from UT-1 cells, a clone of Chinese hamster ovary cells that has markedly elevated reductase activity. This plasmid, designated pRed-10, was identified by differential colony hybridization and hybrid-selected mRNA translation. The mRNA that hybridized to pRed-10 directed the synthesis in vitro of a 90,000-dalton protein that was immunoprecipitated by an antireductase antibody. The same 90,000-dalton protein was immunoprecipitated when UT-1 cells were pulse labeled with [35S]methionine in vivo and rapidly solubilized with boiling NaDodSO4. By blot hybridization, pRed-10 hybridized to mRNAs of 4.2 and 4.7 kilobases in UT-1 cells. Both mRNAs were reduced to undetectable levels when low density lipoprotein, a suppressor of the reductase, was present in the culture medium. These data indicate that the primary translation product of reductase mRNA is a 90,000-dalton protein and that LDL suppresses the reductase in UT-1 cells by drastically reducing the level of its mRNA.
Development of hybridomas secreting monoclonal antibodies to the chicken intestinal 1 alpha,25-dihydroxyvitamin D3 receptor.
Authors: Pike J W , Donaldson C A , Marion S L , Haussler M R
Published: May 2006

Four hybridomas that secrete monoclonal antibodies to the chicken intestinal cytoplasmic 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor have been obtained. Splenic lymphocytes, derived from two male Lewis rats expressing serum antireceptor activity after repeated immunization with a partially purified preparation of this protein, were fused with two nonsecreting murine myeloma cell lines, SP2/0-Ag14 and P3-X63-Ag8.653. Viable hybrids were screened for anti-chicken intestinal 1,25-(OH)2D3 receptor activity by incubation of hybrid media with receptor-hormone complex; this was followed by immunoprecipitation with rabbit anti-rat IgG. Of 1,724 hybridomas assayed by this technique, 4 were positive (2 derived from each animal) for the secretion of an antireceptor immunoglobulin molecule. After cloning by limiting dilution, the cell lines (designated SP2/0-4A5, P3-8C8, SP2/0-8D3, and SP2/0-9A7) were expanded into suspension culture. Antibody-induced alterations in the sedimentation pattern of the native 1,25-(OH)2D3 receptor, coupled with Ouchterlony double-diffusion techniques, indicate that SP2/0-4A5 secretes an IgG2a, SP2/0-9A7 produces an IgG2b, and P3-8C8 secretes an IgG. In contrast, SP2/0-8D3 was found to synthesize an IgM. The monoclonal antibodies react with both occupied and unoccupied chicken intestinal receptor and nuclear receptors, and they crossreact with 1,25-(OH)2D3 receptors from a wide variety of tissue and cultured cell types, including human 1,25-(OH)2D3 receptors. These immunological reagents should prove valuable in the elucidation of the molecular action of 1,25-(OH)2D3.
Photoaffinity labeling with 2-azidoadenosine diphosphate of a tight nucleotide binding site on chloroplast coupling factor 1
Authors: Czarnecki Joseph J. , Abbott Marilyn S. , Selman Bruce R.
Published: May 2006

An analog of ADP containing an azido group at the C-2 position of the purine ring has been synthesized and used as an affinity probe of the membrane-bound coupling factor 1 of spinach chloroplast thylakoid membranes. The 2-azido-ADP inhibited light-induced dark binding of ADP at the tight nucleotide binding site on the thylakoid membranes. The 2-azido-ADP itself bound tightly to the thylakoid membranes, with 1 μM as the concentration giving 50% maximum binding. Tight binding of the analog required the thylakoid membranes to be energized, and the nucleotide remained bound after repeated washings of the membranes. The maximum extent of tight binding of the analog (1,2-1.3 nmol/mg of chlorophyll) was stoichiometric with the known coupling factor 1 content of thylakoid membranes but somewhat higher than that observed for ADP (0.5-0.9 nmol per mg of chlorophyll). Tight binding of 2-azido-ADP was decreased by the simultaneous addition of ADP. UV photolysis of washed thylakoid membranes containing tightly-bound 2-azido-[β-
32
P]ADP resulted in the covalent incorporation of label into the membranes. Isolation of the chloroplast coupling factor 1 from these membranes followed by NaDodSO
4
gel electrophoresis demonstrated that the analog was covalently bound to the β subunit of the coupling factor complex.
Low-calorie diet prevents the development of mammary tumors in C3H mice and reduces circulating prolactin level, murine mammary tumor virus expression, and proliferation of mammary alveolar cells
Authors: Sarkar Nurul H. , Fernandes Gabriel , Telang Nitin T. , Kourides Ione A. , Good Robert A.
Published: May 2006

The effect of carlorie intake on the development of spontaneous mammary tumors in virgin C3H mice was studied. Only about 10% of the mice fed a low-calorie diet [10 kcal/day (1 kcal = 4.184 kJ)] since weaning developed mammary tumors, compared to about 60% of those mice that were reared on high-calorie diets (16 kcal/day or lab chow ad lib). In order to understand the mechanism by which a low-calorie diet decreases the occurrence of mammary tumors in mice, we compared the sex cycle, the amounts of circulating thyroid-stimulating hormone (thyrotropin), growth hormone, and prolactin, the production of type A and B virus particles in the mammary glands, and the morphology of the mammary glands of mice fed low- and high-calorie diets. The amount of serum prolactin and the synthesis of type A and B particles in mammary tissues of mice fed a low-calorie diet was markedly decreased compared to those of age-matched mice fed high-calorie diets. In addition, in young mice fed a low-calorie diet, there were fewer mammary alveolar lesions than in mice fed a high-calorie diet, although the size of the lesions was similar. However, in older mice fed the high-calorie diet, the number and size of these lesions were greater than in the mice raised on the low-calorie diet. The other factors that we studied were not affected by calorie restriction. Our findings suggest that the reduction in serum prolactin level, mammary tumor virus production, and proliferation of mammary alveolar lesions associated with dietary calorie restriction is responsible for lowering the incidence of mammary tumors in mice.
Genetic and biochemical analysis of gonococcal IgA1 protease: cloning in Escherichia coli and construction of mutants of gonococci that fail to produce the activity.
Authors: Koomey J M , Gill R E , Falkow S
Published: May 2006

The biological significance of bacterial extracellular proteases that specifically cleave human IgA1 is unknown. We have prepared a gene bank of gonococcal chromosomal DNA in Escherichia coli K-12 using a cosmid cloning system. Among these clones, we have identified and characterized an E. coli strain that elaborates an extracellular endopeptidase that is indistinguishable from gonococcal IgA1 protease in its substrate specificity and action on human IgA1. Analysis of recombinant plasmids and examination of plasmid-specific peptides in minicells have shown that the IgA1 protease activity in E. coli is associated with expression of a Mr 140,000 peptide. We have isolated IgA1 protease-deficient mutants of Neisseria gonorrhoeae by reintroduction of physically defined deletions of the cloned gene into the gonococcal chromosome by transformation.
Partitioning of bone marrow into stem cell regulatory domains.
Authors: Maloney M A , Lamela R A , Banda M J , Patt H M
Published: May 2006

To examine the hypothesis that bone marrow consists of discrete stem cell regulatory volumes or domains, we studied spleen colony-forming unit (CFU-S) population growth kinetics in unirradiated WBB6F1-W/Wv mice receiving various doses of +/+ bone marrow cells. Assay of femoral marrow CFU-S content in the eight recipient dose groups revealed a family of growth curves having an initial dose-independent exponential phase and a subsequent dose-dependent deceleration phase. CFU-S content at the growth transition (inflection point) was not a simple linear function of inoculum dose but was shown rather to reflect a random distribution of initially seeded donor CFU-S in discrete volumes of recipient bone marrow. The inoculum dose resulting in a mean of 1 CFU-S per bone marrow sampling unit was estimated to be 17 x 10(6) bone marrow cells, corresponding to a total marrow uptake of approximately 5100 CFU-S (based on a seeding efficiency factor of 10%). If we assume single-hit kinetics, it follows that the recipient W/Wv bone marrow may contain approximately 5100 domains in which stem cell proliferation is geared to the density of the stem cell population. When the various inocula were corrected for multiple seeding in a given domain, the mean inflection point per domain was similar and indicative of five or so divisions before departure from exponential growth at approximately 20% of final CFU-S content 8 days after bone marrow injection. The partitioning of bone marrow into highly localized functional units is consistent with the putative regulatory role of short-range interactions between stem cells and essential stromal elements.
Lymphocyte-conditioned medium protects human monocyte-macrophages from cholesteryl ester accumulation.
Authors: Fogelman A M , Seager J , Haberland M E , Hokom M , Tanaka R , Edwards P A
Published: May 2006

Exposure of human monocyte-macrophages to as little as 50 microliters of cultured medium from lymphocytes stimulated by concanavalin A (Con A) resulted in a dramatic decrease in the activities of the low density lipoprotein (LDL) receptor pathway, the LDL-dextran sulfate pathway, and the scavenger receptor pathway. This effect was not seen when the monocyte-macrophages were exposed to culture medium from lymphocytes cultured without Con A or with Con A together with alpha-methyl mannoside or control medium without lymphocytes. The activity of 3-hydroxy-3-methyglutaryl-coenzyme A reductase also decreased in monocyte-macrophages exposed to culture medium from stimulated lymphocytes. Acyl-CoA:cholesterol O-acyltransferase activity, protein synthesis, protein content, phagocytosis of heat-killed yeast, and non-receptor-mediated endocytosis were not inhibited. Monocyte-macrophages exposed to malondialdehyde altered-LDL in the presence of stimulated lymphocyte culture medium accumulated substantially less cholesteryl esters than did cells in control medium. We propose that substances produced by stimulated lymphocytes may be useful in protecting macrophages from cholesteryl ester accumulation.
Expression of a cloned adenovirus gene is inhibited by in vitro methylation.
Authors: Vardimon L , Kressmann A , Cedar H , Maechler M , Doerfler W
Published: May 2006

In many viral and nonviral eukaryotic systems, an inverse correlation has been observed between the extent of DNA methylation at 5'-C-C-G-G-3' sites and the extent of expression of specific genes as mRNA. The E2a region of adenovirus serotype 2 (Ad2) DNA encodes the Ad2-specific DNA binding protein required for viral DNA replication. In three lines of Ad2 transformed hamster cells (HE1, HE2, and HE3), multiple copies of the major part of the Ad2 genome persist in an integrated state. Cell lines HE2 and HE3 do not express the DNA-binding protein whereas line HE1 does so. It has been shown that, in cell line HE1, all 5'-C-C-G-G-3' (Hpa II/MspI) sites in the E2a region remain unmethylated. Conversely, in lines HE2 and HE3 lacking expression of the E2a region all Hpa II sites are methylated. The cloned E2a region of Ad2 DNA, the HindIII A fragment in pBR322, was methylated in vitro by using Hpa II DNA methyltransferase (5'-C-C*G-G-3') or was left unmethylated. In vitro methylation did not break or nick supercoiled circular DNA. Methylated or unmethylated DNA was then microinjected into the nuclei of Xenopus laevis oocytes, and the subsequent synthesis of Ad2-specific RNA was monitored. In vitro-methylated DNA remained in the methylated state for 24 hr on microinjection into nuclei of xenopus oocytes; unmethylated DNA remained unmethylated. When the injected DNA had been methylated by using Hpa H DNA methyltransferase, Ad2-specific RNA was not synthesized as late as 24 hr after microinjection. Unmethylated DNA was readily expressed into Ad2-specific RNA. As an internal control, unmethylated histone genes (h22 DNA) from sea urchin were microinjected together with methylated E2a DNA from Ad2. Ad2-specific RNA was not found; h22 DNA-specific RNA was readily detected. This finding ruled out nonspecific inhibitory effects in the methylated DNA preparation. Ir was also shown that transcription of the unmethylated HindIII A fragment of Ad2 DNA in Xenopus oocytes was initiated on the late promoter of the E2a region. The same promoter was used in productively infected KB cells. Methylation by BsuRI methylse (5'-G'G-C*C-3') did not inactivate the HindIII A fragment. These results provide evidence for the notion that methylated sequences at highly specific sites are involved in the regulation of gene expression. The actual nature of the regulatory signal is not yet understood.
Two alternative mechanisms for initiation of DNA replication forks in bacteriophage T4: priming by RNA polymerase and by recombination.
Authors: Luder A , Mosig G
Published: May 2006

We show that bacteriophage T4 has two alternative mechanisms to initiate DNA replication; one dependent on Escherichia coli RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6), and one dependent on general recombination. Continued DNA synthesis under recombination-defective conditions was sensitive to rifampin, an inhibitor of RNA polymerase. On the other hand, DNA synthesis accelerated in spite of the present of rifampin if recombination occurred.
Effect of methylation on expression of microinjected genes.
Authors: Waechter D E , Baserga R
Published: May 2006

The cloned genes for the simian virus 40 large tumor antigen and for herpes simplex virus (HSV) thymidine kinase (TK) were methylated with EcoRI methylase. The genes were microinjected into the nuclei of TK-deficient (tk-) cells, and expression of the genes was determined by immunofluorescence staining for the simian virus 40 large tumor antigen and by [3H]thymidine incorporation followed by autoradiography for HSV TK. We found that methylation of the simian virus 40 gene, under EcoRI or EcoRI* conditions, resulting in methylation at sites within the gene and in the surrounding sequences, has no effect on expression of the large tumor antigen when the gene is manually microinjected into mammalian nuclei. However, methylation of the HSV tk gene at the two EcoRI sites markedly reduces or abolishes the expression of this gene. One of the EcoRI sites of HSV tk is approximately 1.1 kilobases downstream from the 3' end of the gene and is believed to have no regulatory function in the expression of the tk gene. The other EcoRI site is 79 base pairs upstream from the 5' end of the gene and has considerable homology to the regulatory sequence proposed by [Benoist C., O'Hare, K., Breathnach, R., & Chambon, P. (1980) Nucleic Acids Res. 8, 127-142]. Our results are direct proof that methylation can alter gene expression and also that the effect depends strictly on the sites that are methylated.
Epidermal growth factor and insulin cause normal chicken heart mesenchymal cells to proliferate like their Rous sarcoma virus-infected counterparts.
Authors: Balk S D , Shiu R P , LaFleur M M , Young L L
Published: May 2006

Normal chicken heart mesenchymal cells at low culture density are proliferatively quiescent in a physiological culture medium containing heparinized, heat-inactivated, chicken plasma at 10%. Rous sarcoma virus-infected chicken heart mesenchymal cells, on the other hand, proliferate maximally in this same medium, undergoing a 60-fold increase in cell number during 4 days of exponential growth. When normal heart mesenchymal cells are cultured for 4 days in the presence of epidermal growth factor at 1 micrograms/ml they undergo a 16-fold increase in number, with graded responses to lower concentrations of the factor. In the presence of insulin at 10 micrograms/ml, normal heart mesenchymal cells multiply by a factor of 3 over a 4-day period. The addition of epidermal growth factor (1 microgram/ml) and insulin (10 micrograms/ml) to cultures of normal chicken heart mesenchymal cells causes these cells to proliferate at a rate comparable to that of their RSV-infected counterparts.
Changes in the conformation of influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion.
Authors: Skehel J J , Bayley P M , Brown E B , Martin S R , Waterfield M D , White J M , Wilson I A , Wiley D C
Published: May 2006

A conformational change in the hemagglutinin glycoprotein of influenza virus has been observed to occur to pH values corresponding to those optimal for the membrane fusion activity of the virus. CD, electron microscopic, and sedimentation analyses show that, in the pH range 5.2-4.9, bromelain-solubilized hemagglutinin (BHA) aggregates as protein-protein rosettes and acquires the ability to bind both lipid vesicles and nonionic detergent. Trypsin treatment of BHA in the pH 5.0-induced conformation indicates that aggregation is a property of the BHA2 component and that the conformation change also involves BHA1. The implications of these observations for the role of the glycoprotein in membrane fusion are discussed.
Template-directed pausing in in vitro DNA synthesis by DNA polymerase a from Drosophila melanogaster embryos.
Authors: Kaguni L S , Clayton D A
Published: May 2006

The activity of Drosophila melanogaster DNA polymerase alpha on DNA-primed single-stranded DNA templates has been examined. The DNA templates contain a 1471-nucleotide sequence from the heavy-strand origin region of mouse mtDNA inserted into the single-stranded bacteriophage vector M13Gori1. Preferred sites for pausing of in vitro DNA synthesis have been mapped within the cloned mtDNA insert and in the G4 cDNA strand origin which is contained within the vector DNA. Analysis of nascent DNA strands from replicative intermediates has revealed that pause sites are discrete and lie both at the positions of predicted stable dyads and in regions lacking the potential for formation of such structures. The patterns of kinetic pause sites observed for Escherichia coli DNA polymerase III holoenzyme is qualitatively similar to that found for DNA polymerase alpha. A subset of the observed kinetic pause signals are recognized by E. coli DNA polymerase I under similar conditions.
Molecular cloning and physical mapping of varicella-zoster virus DNA.
Authors: Straus S E , Owens J , Ruyechan W T , Takiff H E , Casey T A , Vande Woude G F , Hay J
Published: May 2006

Varicella-zoster virus (VZV) DNA was cleaved with restriction endonuclease EcoRI, and most of the resulting fragments were successfully cloned in the phage vector lambda gtWES . lambda B. Double digestions of cloned fragments with EcoRI and BamHI and hybridizations to blot-transferred BamHI digests of VZV DNA were used to construct a physical map of the genome. The molecular termini of the DNA were identified by restriction enzyme analysis after exonuclease III digestion. The data indicate that VZV DNA exists in two isomeric forms that differ by inversion of one short terminal genome segment. Electron microscopic studies revealed that the short genome segment consists of a terminal revealed that the short genome segment consists of a terminal sequence of about 3.4 X 10(6) daltons that is separated from an internal inverted repeat of itself by a 5.8 X 10(60)-dalton unique DNA segment.
Effects of DNA base analogs on transcription termination at the tryptophan operon attenuator of
Escherichia coli
Authors: Farnham Peggy J. , Platt Terry
Published: May 2006

We have devised a method to specifically incorporate deoxyribonucleotide base analogs
in vitro
into either strand of the tryptophan (
trp
) operon attenuator region, using primed synthesis on bacteriophage M13 derivatives carrying cloned
trp
attenuator DNA. We have employed these techniques to extend previous studies implicating both RNA-RNA and RNA-DNA interactions in transcription termination in an attempt to determine the nature of the contribution from the template DNA molecule in termination regions. In general, we find that the dramatic effects upon transcription termination seen with base analog incorporation into mRNA do not occur when similar analogs are incorporated into the DNA. Only the analog 2,6-diaminopurine deoxyribonucleotide triphosphate (dDapTP), which strengthens A·T or A·U base pairing, elicits a significant response: in the template DNA strand, the presence of this analog increases read-through at the
trp
attenuator. The analog 5-bromouracil deoxyribonucleoside triphosphate (BrdUTP), which also strengthens pairing with its complementary base, has no detectable effect on termination when it is placed in either strand of the
trp
attenuator or the mutant attenuator
trp a1419
. Surprisingly, though the analog 5-iodocytosine deoxyribonucleoside triphosphate (IdCTP) does not affect termination, it has a great effect on initiation of transcription, depressing
trp
promoter activity as well as stimulating transcription from other regions. These results support the postulated interaction between terminal uridines in mRNA and the template DNA strand in enhancing termination and suggest that there are no significant additional contributions from the DNA. In addition, the novel use of M13 derivatives for incorporating analogs into the DNA on a preparative scale provides a technique for introducing mutations in a general but controlled fashion as a new means for studying other regulatory regions.
Molecular cloning of murine endogenous viral sequences and expression of a newly constructed recombinant murine leukemia virus DNA in transfected mink cells.
Authors: van der Hoorn F A , Onnekink C , van der Putten H , Zijlstra M , Bloemers H P
Published: May 2006

In the process of molecularly cloning unintegrated proviral DNA from NIH-3T3 mouse cells infected with Rauscher murine leukemia virus, we observed the occurrence of clones with inserts smaller than the expected Rauscher murine leukemia virus fragments. The insert of one of these clones, lambda.Xe-1, was characterized in more detail. It had a size of 3.5 kilobases. The restriction map was similar but not identical to that of the envelope regions of Moloney and Rauscher murine leukemia viruses. After ligation to previously cloned Moloney murine leukemia viral sequences and transfection of the ligated DNA into mink lung cells a nondefective xenotropic murine leukemia virus, XH-19, was isolated. Restriction mapping of proviral DNA isolated from mink lungs cells chronically infected with XH-19 showed the presence of Moloney murine leukemia virus-derived sequences coupled to xenotropic viral sequences.
Membrane potential changes induced by the ouabain-like compound extracted from mammalian brain.
Authors: Lichtstein D , Samuelov S
Published: May 2006

The electrical membrane potential (delta psi) of chicken embryo fibroblasts in tissue culture was determined to be -30.5 +/- 2.9 mV as measured by distribution of the lipophilic [3H]tetraphenylphosphonium cation (Ph4P+). Stimulation of the electrogenic activity of the Na+,K+-ATPase by the ionophore monensin induces a hyperpolarization of approximately 47 mV and a new delta psi of -77.3 +/- 5.7 mV. The effects of the cardiac glycoside ouabain and an "ouabain-like compound" (OLC), which was extracted and partially purified from sheep brain, were contrasted using both the resting and hyperpolarized fibroblasts. Addition of OLC or ouabain to the incubation medium for short periods of time does not alter the cells' resting delta psi. However, OLC and ouabain block monensin-induced hyperpolarization. The inhibitory effects of OLC, like ouabain, are dose dependent, with half-maximal inhibition occurring at an amount of OLC equivalent to that found in 1.6 g of brain (wet weight) per ml and at 0.85 microM ouabain. In addition, the maximal actions of ouabain and OLC are not additive. These results show that the endogenous OLC specifically affects the delta psi of intact cells by a mechanism analogous to that of ouabain--i.e., inhibition of the Na+,K+-ATPase.
Trophic and size structure of West Indian bird communities
Authors: Faaborg John
Published: May 2006

Much discussion has occurred in recent years on whether observed patterns of structure in island bird communities are the result of competitive interactions among species or independent rates of colonization and extinction. Here two patterns of structure are presented for birds on 12 West Indian islands. Each of four major foraging guilds shows a distinct species-area pattern on the islands and saturation of species within habitats on larger islands. Frugivores have the steepest species-area curve and highest species numbers at saturation while nectarivores are lowest in both values. Coexisting guild members in saturated habitats are generally of different sizes, with weight differences by a factor of 2 common. On smaller islands, small guild members are absent and size differences among coexisting guild members may increase. In many cases, birds have apparently shifted size to conform to the structural patterns. The consistency of the patterns and variation within component species is highly compatible to explanations invoking competition and complements previously described population and community characteristics of West Indian birds.
Rejection of stable cultured allografts by active or passive (adoptive) immunization.
Authors: Vesole D H , Dart G A , Talmage D W
Published: May 2006

Injection of live lymphoid cells of donor strain or immune cells of recipient strain resulted in rejection of previously stable cultured mouse thyroid allografts. The results are interpreted to indicate that a cultured graft is relatively ineffective in activating recipient lymphocytes but is capable of maintaining them in an activated state and serving as a target for them once they are activated.
Reaction mechanism of mRNA guanylyltransferase from rat liver: isolation and characterization of a guanylyl-enzyme intermediate.
Authors: Mizumoto K , Kaziro Y , Lipmann F
Published: May 2006

Rat liver RNA guanylyltransferase catalyzes a GTP-PPi exchange reaction in the absence of acceptor RNA [Mizumoto, K. & Lipmann, F. (1979) Proc. Natl. Acad. Sci. USA 76, 4961-4965] suggesting that the reaction proceeds through the formation of a covalent guanylylated intermediate. We now present more direct evidence for the existence of the enzyme-GMP intermediate: (i) the enzyme-[32P]GMP intermediate was formed on incubation of rat liver guanylyltransferase with [alpha-32P]GTP and migrated as a single radioactive band with Mr 69,000 on NaDodSO4/polyacrylamide gel electrophoresis, and (ii) the intermediate isolated on gel filtration can transfer its GMP moiety to ppGpCpC-poly(A2,U2,G) to form the capped RNA molecule or it can react with PPi to regenerate GTP. The formation of the intermediate was dependent on Mg2+ and was strongly inhibited by PPi. The addition of pyrophosphatase markedly increased the amount of the intermediate complex. On blue dextran-Sepharose affinity column chromatography, the activity of guanylyltransferase to form an enzyme-[32P]GMP intermediate comigrated with activities of cap formation and GTP-PPi exchange. A phosphoamide type linkage between GMP and enzyme is suggested by its acidlabile and alkali-stable nature and also by the susceptibility to acidic hydroxylamine. These results indicate that the reaction catalyzed by rat liver guanylyltransferase occurs through the following two partial steps: (i) E + GTP in equilibrium E-pG + PPi; and (ii) E-pG + ppN .....leads to GpppN .....+ E.
Isolation and identification of a cDNA clone corresponding to an HLA-DR antigen beta chain.
Authors: Wiman K , Larhammar D , Claesson L , Gustafsson K , Schenning L , Bill P , Böhme J , Denaro M , Dobberstein B , Hammerling U , Kvist S , Servenius B , Sundelin J , Peterson P A , Rask L
Published: May 2006

The HLA-D locus in the major histocompatibility complex controls the expression of the genetically polymorphic HLA-DR antigens. mRNA coding for the beta chains of these antigens was partially purified from the human lymphoblastoid cell line Raji. The mRNA was copied into double-stranded cDNA and cloned in Escherichia coli. One clone, pDR-beta-1, obtained by hybrid selection, carries a 1070-base-pair insert comprising all of the coding region except the signal sequence and a substantial portion of the untranslated region. To identify pDR-beta-1, highly purified HLA-DR antigen beta chains derived from Raji cells were subjected to NH2-terminal amino acid sequence determination. This sequence displayed extensive homology with that deduced from the nucleotide sequence at the 5' end of the pDR-beta-1 coding region. Taken together, the amino acid and nucleotide sequences strongly argue in favor of Raji cells containing at least two beta-chain loci.
Specificity of receptor-mediated recognition of malondialdehyde-modified low density lipoproteins.
Authors: Haberland M E , Fogelman A M , Edwards P A
Published: May 2006

Blood-borne human monocytes and macrophages derived from human monocytes in vitro express an active low density lipoprotein (LDL) receptor and an active receptor for negatively charged proteins, the scavenger receptor. When less than 15% of the lysine residues of human LDL were modified by malondialdehyde while the lipoprotein was in solution, recognition and uptake of the modified lipoprotein occurred via the LDL receptor. Further modification resulted in threshold recognition and uptake by the scavenger receptor with concomitant loss of recognition by the LDL receptor. The rate of degradation via the LDL receptor pathway was inversely related to the degree of modification whereas that mediated by the scavenger receptor was independent of the extent of incorporation of malondialdehyde once threshold recognition was achieved. In contrast to the interaction of LDL with malondialdehyde in solution, modification of less than 15% of the lysine residues of LDL adsorbed to heparin-Sepharose resulted in recognition and uptake by the scavenger receptor. The scavenger receptor-mediated uptake of malondialdehyde-modified LDL may be dependent on formation of recognition sites involving specific modified lysine residues or changes in the conformation of LDL induced by neutralization of specific lysine residues of the apoB polypeptides or both.
Liposome-mediated delivery of tobacco mosaic virus RNA into tobacco protoplasts: A sensitive assay for monitoring liposome-protoplast interactions
Authors: Fraley Robert T. , Dellaporta Stephen L. , Papahadjopoulos Demetrios
Published: May 2006

Tobacco mosaic virus (TMV) RNA was encapsulated in large, unilamellar phospholipid vesicles (liposomes), and the encapsulated TMV RNA was shown to be infectious when incubated with tobacco protoplasts under appropriate conditions. Maximal virus production in protoplasts was observed after their incubation with TMV RNA entrapped in phosphatidylserine/cholesterol liposomes. Infection was dependent on the presence of polyalcohols in the incubation mixture. Other parameters, such as the extent of vesicle binding, the cell-induced leakage of vesicle contents, and the degree of liposome toxicity were shown to be important in determining the efficiency of infectivity. Liposome-mediated delivery offers an efficient and reproducible method for introducing RNA into plant protoplasts.
Characterizing harmonic immersions of surfaces with indefinite metric
Authors: Milnor Tilla Klotz
Published: May 2006

Harmonic maps
X
:(
S,h
) →
N
from a 2-manifold
S
with indefinite metric
h
to a semi-Riemannian manifold
N
are characterized, assuming that the induced metric I is nondegenerate. Except in one very special case, the characterizations involve a canonically determined holomorphic quadratic differential on a naturally chosen conformal structure. This is surprising because the Euler-Lagrange equation that
X
must satisfy is basically the wave equation. The Gauss map of a spacelike or timelike surface in Minkowski 3-space is shown to be harmonic if and only if mean curvature is constant. Finally, it is noted that a harmonic map
X
:(
S,h
) →
N
with indefinite
h
and nondegenerate I normally gives rise to a sine-Gordon, a sinh-Gordon, or a cosh-Gordon equation, provided that the intrinsic curvature of I is constant.