bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipid-derived signals that discriminate wound- and pathogen-responsive isoprenoid pathways in plants: methyl jasmonate and the fungal elicitor arachidonic acid induce different 3-hydroxy-3-methylglutaryl-coenzyme A reductase genes and antimicrobial isoprenoids in Solanum tuberosum L.
Authors: Choi D , Bostock R M , Avdiushko S , Hildebrand D F
Published: May 2006

Induction of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR; EC 1.1.1.34) is essential for the synthesis of steroid derivatives and sesquiterpenoid phytoalexins in solanaceous plants following mechanical injury or pathogen infection. Gene-specific probes corresponding to different HMGR genes (hmg1 and hmg2) were used to study HMGR expression in potato tissue following treatment with methyl jasmonate, a lipoxygenase product of linolenic acid, or arachidonic acid, an elicitor present in the lipids of the potato late blight fungus Phytophthora infestans. Treatment of potato discs (2.2 cm in diameter) with low concentrations (0.45-45 nmol per disc surface) of methyl jasmonate nearly doubled the wound-induced accumulation of hmg1 transcripts and steroid-glycoalkaloid (SGA) accumulation, reduced the abundance of hmg2 transcripts, and did not induce phytoalexins. High concentrations of methyl jasmonate (2-4.5 mol per disc surface) suppressed hmg1 mRNA and SGA accumulation but did not affect hmg2 mRNA abundance or induce phytoalexins. In contrast, arachidonate treatment strongly suppressed hmg1 and strongly induced hmg2 mRNA in a concentration-dependent manner. There was a corresponding suppression of SGA accumulation and an induction of sesquiterpene phytoalexin accumulation by this elicitor. Lipoxygenase inhibitors reduced the wound-induced accumulation of hmg1 transcripts and suppressed SGA levels, effects that were overcome by exogenous methyl jasmonate (45 nmol per disc surface). The results (i) suggest that methyl jasmonate can function as a signal for hmg1 expression and SGA induction following wounding and (ii) indicate that the arachidonate- and jasmonate-response pathways are distinct in relation to HMGR gene expression and isoprenoid product accumulation. The results also are consistent with placement of the HMGR activities encoded by hmg1 and hmg2 within discrete steroid and sesquiterpenoid biosynthetic channels.
Vaccines for bacterial sexually transmitted infections: a realistic goal?
Authors: Sparling P F , Elkins C , Wyrick P B , Cohen M S
Published: May 2006

Bacterial infections of the genital tract (gonorrhea, chlamydia, chancroid, syphilis) are common and cause significant morbidity. Their importance is heightened by recent appreciation of their roles in facilitation of transmission of the human immunodeficiency virus (HIV). Each is capable of causing repeated infections, suggesting lack of permanent broadly effective immunity. An effective vaccine has yet to be developed for any of these diseases. Rapid progress in understanding the molecular basis for pathogenesis of infection, including mechanisms for escape from otherwise effective immune surveillance and mechanisms for causing injury to host cells, has stimulated renewed efforts to make vaccines for some of these infections. Progress has been greatest for Neisseria gonorrhoeae and Chlamydia trachomatis. Present emphasis is on the major or principal outer membrane proteins of N. gonorrhoeae and C. trachomatis, based on evidence for neutralizing antibodies directed against surface-exposed variable domains of each of these proteins. Other surface-exposed proteins, including the iron-repressible transferrin receptor in gonococci and certain heat-shock proteins in chlamydia, also may be targets for vaccines. Although much remains to be learned, cautious optimism is warranted.
Blocking of retroviral infection at a step prior to reverse transcription in cells transformed to constitutively express interferon beta.
Authors: Vieillard V , Lauret E , Rousseau V , De Maeyer E
Published: May 2006

We are developing methods for somatic-cell gene therapy directed against infection with human immunodeficiency virus, by enhancing antiviral resistance of target cells through the constitutive production of autocrine interferon (IFN). Using the human IFN-beta coding sequence under the constitutive low-expression control of a 0.6-kb murine H-2Kb promoter-fragment, we have constructed a retroviral vector, HMB-KbHuIFN beta, and have transformed cells of the T98G human neuroblastoma line, the U-937 human promonocytic line, and the CEM human lymphocytic line. These human IFN-beta-transformed cell populations have acquired a low, constitutive production of human IFN, while replicating at a rate similar to that of untransformed cells and of cells transformed with the control vector carrying a human IFN-beta sequence encoding an inactive, mutated protein. In the three different cell populations tested, transformation with the HMB-KbHuIFN beta vector resulted in a 1.3-2.3 log10 reduction in the number of cells infected with a defective amphotropic MFG-LaZ retrovirus. A kinetic study of the fate of the MFG-LacZ retrovirus in the culture medium and intracellularly immediately after exposure of the cells to virus revealed a significant reduction of the appearance of intracellular virus in human IFN-beta-transformed cells. A similar effect was obtained by treating untransformed T98G, U-937, and CEM cells with exogenous human IFN-beta. The blocking effect of autocrine or exogenous human IFN-beta on viral entry was not limited to virus specific for the amphotropic receptor but was also obtained in murine IFN-beta-treated NIH 3T3 mouse fibroblasts infected with an ecotropic MFG-LacZ retrovirus. Infection of human IFN-beta-transformed CEM cells with human immunodeficiency virus type 1 gave comparable results. Immediately following exposure of the cells to human immunodeficiency virus, a kinetic study of the fate of the virus failed to reveal the appearance of intracellular virus and showed that the majority of the input virus remained in the extracellular medium. We conclude that low autocrine IFN-beta synthesis, or exposure of cells to exogenous IFN-beta, prevents virus from getting inside the cells, regardless of the virus receptor involved.
A family of cellular proteins related to snake venom disintegrins.
Authors: Weskamp G , Blobel C P
Published: May 2006

Disintegrins are short soluble integrin ligands that were initially identified in snake venom. A previously recognized cellular protein with a disintegrin domain was the guinea pig sperm protein PH-30, a protein implicated in sperm-egg membrane binding and fusion. Here we present peptide sequences that are characteristic for several cellular disintegrin-domain proteins. These peptide sequences were deduced from cDNA sequence tags that were generated by polymerase chain reaction from various mouse tissue and a mouse muscle cell line. Northern blot analysis with four sequence tags revealed distinct mRNA expression patterns. Evidently, cellular proteins containing a disintegrin domain define a superfamily of potential integrin ligands that are likely to function in important cell-cell and cell-matrix interactions.
Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails.
Authors: Charo I F , Myers S J , Herman A , Franci C , Connolly A J , Coughlin S R
Published: May 2006

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine family of cytokines that mediate leukocyte chemotaxis. The potent and specific activation of monocytes by MCP-1 may mediate the monocytic infiltration of tissues in atherosclerosis and other inflammatory diseases. We have isolated cDNAs that encode two MCP-1-specific receptors with alternatively spliced carboxyl tails. Expression of the receptors in Xenopus oocytes conferred robust mobilization of intracellular calcium in response to nanomolar concentrations of MCP-1 but not to related chemokines. The MCP-1 receptors are most closely related to the receptor for the chemokines macrophage inflammatory protein 1 alpha and RANTES (regulated on activation, normal T expressed and secreted). The identification of the MCP-1 receptor and cloning of two distinct isoforms provide powerful tools for understanding the specificity and signaling mechanisms of this important chemokine.
Nerve growth factor and its low-affinity receptor promote Schwann cell migration.
Authors: Anton E S , Weskamp G , Reichardt L F , Matthew W D
Published: May 2006

Migrating Schwann cells in developing or regenerating peripheral nerves are known to express dramatically increased levels of nerve growth factor (NGF) and the low-affinity NGF receptor (LNGFR). Schwann cells do not express detectable pp140trk, the NGF-activated receptor tyrosine kinase which is essential for neuronal responses to NGF. The temporal correlation observed in Schwann cells between migration and the enhanced expression of NGF and LNGFR suggests that NGF and LNGFR may promote Schwann cell migration. To test this possibility, we examined the effects of NGF on Schwann cell migration on cryostat sections of biologically relevant NGF-poor and NGF-rich substrates--normal or denervated peripheral (sciatic) nerve, untreated or pretreated with NGF. Results show that Schwann cells migrate more rapidly on denervated than on normal sciatic nerve. Antibodies to NGF or to LNGFR strongly, but incompletely, inhibit enhanced migration on denervated nerves. Pretreatment of denervated nerve sections with NGF increases further the rate of Schwann cell migration. The same antibodies to NGF or to LNGFR abolish this response. These results suggest that one function of the elevated levels of NGF known to be present in embryonic and regenerating peripheral nerves is to promote the migration of Schwann cells. In contrast to neurons, where pp140trk appears to be the functionally critical NGF receptor, NGF responses in Schwann cells depend on LNGFR.
Antigen-binding glycosylation inhibiting factor from a human T-cell hybridoma specific for bee venom phospholipase A2.
Authors: Gomi H , Tagaya Y , Nakano T , Mikayama T , Ishizaka K
Published: May 2006

We obtained human T-cell hybridomas that are specific for bee venom phospholipase A2 (PLA2) and constitutively secrete glycosylation inhibiting factor (GIF). Upon crosslinking of CD3, the hybridoma produced GIF having affinity for PLA2. When affinity-purified PLA2-binding GIF was used as an immunogen, monoclonal antibodies specific for the antigen-binding GIF were obtained. Monoclonal antibody 110BH3 bound the antigen-binding GIF but failed to bind the 13-kDa nonspecific GIF, as determined by both bioassay and ELISA. In contrast, 388F1, a monoclonal antibody against nonspecific GIF, gave ELISA signals with both the nonspecific GIF and the antigen-binding GIF. Gel filtration of affinity-purified antigen-binding GIF revealed the presence of a 72- to 80-kDa protein which gave ELISA signals with both 110BH3 and 388F1 and contained GIF bioactivity. Upon reduction and alkylation, the antigen-binding GIF dissociated into a 62- to 64-kDa protein which gave positive ELISA with antibody 110BH3 but no signal with antibody 388F1, and a 15-kDa protein, which gave ELISA signal with the 388F1 but not with 110BH3. Immunoblotting of a PLA2-binding GIF preparation revealed that under reducing conditions, the antigen-binding GIF dissociated a 13-kDa peptide which reacted with polyclonal antibodies against recombinant GIF. The results indicate that the 13-kDa nonspecific GIF is a subunit of antigen-binding GIF. The PLA2-binding GIF has affinity for an epitope, representing amino acid residues 19-28 in PLA2 which appears to be an external structure in the antigen.
Intracellular calcium dependence of gene expression in single T lymphocytes.
Authors: Negulescu P A , Shastri N , Cahalan M D
Published: May 2006

In T lymphocytes, intracellular Ca2+ concentration ([Ca2+]i) rises within seconds of T-cell antigen-receptor stimulation and initiates the synthesis and secretion of interleukin 2, a cytokine essential for T-cell proliferation and the immune response. Using video-imaging techniques, we tracked [Ca2+]i signals in individual T cells and measured subsequent expression of a beta-galactosidase reporter gene (lacZ) controlled by the NF-AT element of the interleukin 2 enhancer. [Ca2+]i spikes elicited by monoclonal antibody binding to the CD3 epsilon subunit of the T-cell receptor were positively correlated with gene expression, but varied widely between individual cells and were therefore difficult to relate quantitatively to lacZ expression. The [Ca2+]i dependence of NF-AT-regulated gene expression was determined by elevating [Ca2+]i with either thapsigargin or ionomycin and then "clamping" [Ca2+]i to various, stable levels by altering either extracellular [Ca2+] or extracellular [K+]. Raising [Ca2+]i from resting levels of 70 nM to between 200 nM and 1.6 microM increased the fraction of cells expressing lacZ, with Kd approximately 1 microM. Activation of protein kinase C enhanced the [Ca2+]i sensitivity of gene expression (Kd = 210 nM), whereas stimulation of protein kinase A inhibited [Ca2+]i-dependent gene expression. The experiments described here provide single-cell measurements linking a second messenger to gene expression in individual cells.
Nervous control of juvenile hormone biosynthesis in Locusta migratoria.
Authors: Horseman G , Hartmann R , Virant-Doberlet M , Loher W , Huber F
Published: May 2006

In Locusta migratoria migratorioides R. and F., two types of brain neurons innervate the juvenile hormone (JH)-producing corpora allata (CA). Thirteen cells in each pars lateralis (PL) innervate the ipsilateral CA, while four cells (two in each PL) innervate both glands. We investigated possible influences of these two neuronal types on JH production by a newly developed method. A radiochemical assay was used to measure hourly JH production by a CA with intact nerve connections to the brain. Then, changes in hormone production due to selective nerve stimulation or transection were assessed. In control preparations JH production per h remained approximately constant for at least 9 h. Simultaneous electrical stimulation of all neurons innervating one CA (i.e., 13 ipsilateral plus 4 bilaterally innervating cells) always inhibited JH production, while their transection led to a rapid progressive increase in JH biosynthesis in CA from females with oocytes longer than 4.5 mm. Thus, there is strong neurally mediated inhibition of the CA at certain phases of the vitellogenic cycle. The dramatic effects of nerve transection show that in vitro rates of JH production are an unreliable indicator of in vivo levels. Selective stimulation of the four neurons innervating both CA suggests that they do modulate JH biosynthesis but the effect varies qualitatively depending on the phase of the vitellogenic cycle.
Yeast farnesyl-diphosphate synthase: site-directed mutagenesis of residues in highly conserved prenyltransferase domains I and II.
Authors: Song L , Poulter C D
Published: May 2006

Prenyltransferases that catalyze the fundamental chain elongation reaction in the isoprenoid biosynthetic pathway contain several highly conserved amino acids, including two aspartate-rich regions thought to be involved in substrate binding and catalysis. We report a study of site-directed mutants for yeast farnesyl-diphosphate synthase (FPPSase; geranyl-diphosphate:isopentenyl-diphosphate, EC 2.5.1.10), a prenyltransferase that catalyzes the sequential 1'-4 coupling of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate and geranyl diphosphate. A recombinant form of FPPSase extended by a C-terminal -Glu-Glu-Phe alpha-tubulin epitope (EEF in single-letter amino acid code) was engineered to facilitate rapid purification of the enzyme by immunoaffinity chromatography and to remove traces of contaminating activity from wild-type FPPSase in the Escherichia coli host. Ten site-directed mutants were constructed in FPPSase::EEF. The six aspartates in domain I (at positions 100, 101, and 104) and domain II (at positions 240, 241, and 244) were changed to alanine (mutants designated D100A, D101A, D104A, D240A, D241A, and D244A); three arginine residues were changed, Arg-109 and Arg-110 to glutamine and Arg-350 to alanine (mutants designated R109Q, R110Q, and R350A); and Lys-254 was converted to alanine (mutant designated K254A). Mutations of the aspartatic residues and nearby arginine residues in domain I and Asp-240 and Asp-241 in domain II drastically lowered the catalytic activity of FPPSase::EEF. The D244A and K254A mutants were substantially less active, while kcat and the Michaelis constants for the R350A mutant were similar to those of FPPSase::EEF. Addition of an -EEF epitope to the C terminus of wild-type FPPSase resulted in a 14-fold increase of KmIPP and a 12-fold decrease of kcat, suggesting that the conserved hydrophilic C terminus of the enzyme may have a role in substrate binding and catalysis.
Enhanced DNA sequencing by hybridization.
Authors: Broude N E , Sano T , Smith C L , Cantor C R
Published: May 2006

An enhanced version of DNA sequencing by hybridization (SBH), termed positional SBH (PSBH), has been developed. PSBH uses duplex probes containing single-stranded 3' overhangs, instead of simple single-stranded probes. Stacking interactions between the duplex probe and a single-stranded target should provide enhanced stringency in distinguishing perfectly matched 3' sequences. A second enhancement is the use of enzyme-catalyzed steps, instead of pure physical hybridization. The feasibility of this scheme has been investigated using biotinylated duplex probes containing single-stranded 5-base 3' overhangs, immobilized on streptavidin-coated magnetic beads. Ligation of a single-stranded target, hybridized to the single-stranded region of the duplex probes, provided enhanced discrimination of perfectly matched targets from those containing mismatches. In distinction to the serious complications caused by base composition effects in ordinary SBH, there was little effect of base composition in PSBH. The hardest mismatch to discriminate was the one furthest from the phosphodiester bond formed by ligation. However, mismatches in this position were efficiently discriminated by 3' extension of the duplex probe using a template-dependent DNA polymerase. These results demonstrate that PSBH offers considerable promise to facilitate actual implementations of SBH.
G-->A hypermutation of the human immunodeficiency virus type 1 genome: evidence for dCTP pool imbalance during reverse transcription.
Authors: Vartanian J P , Meyerhans A , Sala M , Wain-Hobson S
Published: May 2006

The quasispecies model for RNA viruses predicts the existence of a replication error threshold beyond which there is a melting or total loss of sequence information. Retroviral G-->A hypermutation is probably an example. Here it is shown that G-->A transitions may occur in both GpG and GpA dinucleotide contexts. Transitions in GpG preferentially occur via base mispairing at the ends of runs of G residues, whereas G-->A transitions within GpA may result from temporary dislocation of the primer and template strands by a single base. The two circumstances may be related by the local dCTP substrate concentration. An in vitro elongation assay shows that primer/template dislocation is more frequent for the human immunodeficiency virus type 1 reverse transcriptase than for murine or avian retroviral enzymes. Taken together these data suggest that G-->A hypermutation is an example of induced mutation whereby the viral reverse transcriptase is forced into making errors by imbalances in the intracellular dCTP concentration.
Nuclear import of Agrobacterium VirD2 and VirE2 proteins in maize and tobacco.
Authors: Citovsky V , Warnick D , Zambryski P
Published: May 2006

Previously, we have shown that Agrobacterium-plant cell transferred DNA (T-DNA) transport into the host cell nucleus is likely mediated by two specific bacterial proteins, VirD2 and VirE2. Here, we used these proteins to study molecular pathways of nuclear import. First, the role of VirE2 nuclear localization signals (NLSs) in the T-DNA transport pathway was examined by using tobacco plants transgenic for deletion mutants of VirE2. In these plants, the virulence of wild-type Agrobacterium was reduced possibly by competition for the cellular nuclear import machinery. Second, we analyzed the nuclear localization of VirE2 and VirD2 in the nonhost monocot maize. Part of the known recalcitrance of monocots to transformation by Agrobacterium could be due to a potential selectivity in nuclear import pathways in monocotyledonous and dicotyledonous plants. Nuclear transport of VirD2 and VirE2 in maize leaves and roots was compared to that in tobacco protoplasts and roots. Both proteins accumulated in maize leaf and tobacco protoplast nuclei as well as in nuclei of immature root cells. In contrast, VirD2 and VirE2 expressed in mature roots of maize and tobacco remained cytoplasmic. Point mutations of VirE2 nuclear localization signals, NSE 1 and NSE 2, also revealed that, in maize, the NSE 1 signal was mainly responsible for nuclear import; in contrast, both signals functioned independently in tobacco protoplasts.
Selective inhibition of inducible cyclooxygenase 2 in vivo is antiinflammatory and nonulcerogenic.
Authors: Masferrer J L , Zweifel B S , Manning P T , Hauser S D , Leahy K M , Smith W G , Isakson P C , Seibert K
Published: May 2006

We have examined the role of cyclooxygenase 2 (COX-2) in a model of inflammation in vivo. Carrageenan administration to the subcutaneous rat air pouch induces a rapid inflammatory response characterized by high levels of prostaglandins (PGs) and leukotrienes in the fluid exudate. The time course of the induction of COX-2 mRNA and protein coincided with the production of PGs in the pouch tissue and cellular infiltrate. Carrageenan-induced COX-2 immunoreactivity was localized to macrophages obtained from the fluid exudate as well as to the inner surface layer of cells within the pouch lining. Dexamethasone inhibited both COX-2 expression and PG synthesis in the fluid exudate but failed to inhibit PG synthesis in the stomach. Furthermore, NS-398, a selective COX-2 inhibitor, and indomethacin, a nonselective COX-1/COX-2 inhibitor, blocked proinflammatory PG synthesis in the air pouch. In contrast, only indomethacin blocked gastric PG and, additionally, produced gastric lesions. These results suggest that inhibitors of COX-2 are potent antiinflammatory agents which do not produce the typical side effects (e.g., gastric ulcers) associated with the nonselective, COX-1-directed antiinflammatory drugs.
Recognition of oxidatively damaged erythrocytes by a macrophage receptor with specificity for oxidized low density lipoprotein.
Authors: Sambrano G R , Parthasarathy S , Steinberg D
Published: May 2006

Macrophages specifically bind and internalize oxidatively modified low density lipoprotein (LDL) via the acetyl-LDL receptor and possibly one or more additional receptors jointly designated here as scavenger receptors. It is well accepted that these receptors are intimately involved in the formation of foam cells during atherogenesis. However, the normal physiological or pathophysiological role for these receptors has not been established. Oxidation of plasma membranes is a common accompaniment of cell damage and senescence. In particular, aged erythrocytes demonstrate peroxidation of their cell membrane lipids. In the present studies we show that oxidized human erythrocytes (treated with copper plus ascorbate or hydrogen peroxide) are bound and phagocytosed by mouse peritoneal macrophages in the absence of opsonizing antibodies. There was little or no binding of untreated erythrocytes. Oxidized LDL, but not acetylated or native LDL, inhibited this binding and uptake of oxidized erythrocytes. Inhibitors of scavenger receptor binding, including polyinosinic acid and fucoidin, also prevented binding of the oxidized red blood cells. We suggest that oxidative damage of erythrocytes results in the formation of lipid-protein conjugate(s) closely related to some of the conjugates found in oxidized LDL, making the oxidized erythrocyte a ligand for the macrophage scavenger receptors, apparently at a site distinct from that responsible for the binding of acetylated LDL. Oxidative modification of plasma membranes may represent a general mechanism that marks damaged cells for phagocytosis by macrophages.
Effect of increasing doses of angiotensin II infused into normal and hypertensive Wistar rats on low density lipoprotein and fibrinogen uptake by aortic walls.
Authors: Cardona-Sanclemente L E , Medina R , Born G V
Published: May 2006

The effect of 6 days' s.c. infusions of angiotensin II at increasing doses was determined on the uptake of rat or human low density lipoprotein (LDL) and of human fibrinogen by aorta in normal and spontaneously hypertensive rats. Rat or human LDL or human fibrinogen was injected i.v. 5 days after the start of infusion, and 24 hr later the radioactivity of aortic walls was determined. Body weight was almost constant in control rats and moderately decreased in a dose-dependent way by angiotensin II. Diastolic blood pressure decreased slightly over 6 days in control rats and increased transiently at the lowest dose of angiotensin II and progressively with two higher concentrations. All three angiotensin II concentrations significantly increased the uptake of rat and human LDL and of fibrinogen by aorta. The increase was dose related for rat LDL but not for human LDL or fibrinogen. In spontaneously hypertensive rats of the same age in which blood pressure was higher than in angiotensin II-infused rats, protein uptakes were not increased. The blood content of aortic walls was negligible and not altered by angiotensin II. Therefore, the uptake of atherogenic plasma proteins by rat aorta is increased by angiotensin II, but this effect may be independent of its pressor action.
Magnetic resonance microscopy of mouse embryos.
Authors: Smith B R , Johnson G A , Groman E V , Linney E
Published: May 2006

The increased use of the mouse as a model for various aspects of mammalian biology has caused a renewed interest in developing strategies for examining and comparing normal and abnormal mouse embryonic development and anatomy. In this study, we have explored the use of magnetic resonance microscopy as a tool for these purposes. Techniques for the fixation, embedding, perfusion, and image acquisition of mouse embryos are described. The perfusion of bovine serum albumin-diethylenetriamine pentaacetic anhydride-gadolinium as a contrast agent enhances images of the developing embryonic vasculature during critical stages of organogenesis and allows for comparisons when embryos have been treated with teratogens such as retinoic acid. The acquired three-dimensional data sets are available for archiving, distributing, and postacquisition manipulations such as computer segmentation of anatomical structures.
Rodent BC1 RNA gene as a master gene for ID element amplification.
Authors: Kim J , Martignetti J A , Shen M R , Brosius J , Deininger P
Published: May 2006

ID elements are short interspersed repetitive DNA elements (SINEs) which have amplified in rodent genomes via retroposition, a process involving an RNA intermediate. BC1, an abundant ID-related transcript, is transcribed from a conserved, single-copy gene in rodents. The gene encoding BC1 RNA represents one of the earliest and possibly the first ID-containing sequence. Comparison of consensus sequences of each rodent ID with its corresponding BC1 RNA gene showed that the variations of BC1 RNA within rodents corresponded to specific changes within the ID consensus sequence for each rodent species. This supports the hypothesis that the BC1 gene is a master gene responsible for the amplification and evolution of ID elements. The rat ID family consists of at least four subfamilies, with the oldest subfamily having been derived from the BC1 RNA. The other three subfamilies appear to have been derived from a new master gene(s), which has been responsible for the large increase in ID element copy number within the rat genome. We have found that the guinea pig genome contains two copies of the BC1 gene, apparently the result of a DNA-mediated duplication event. Both of these guinea pig BC1 genes have a conserved TATA-like element in the 5' flanking region and have contributed to guinea pig ID amplifications.
Targeted gene duplication and disruption for analyzing quantitative genetic traits in mice.
Authors: Smithies O , Kim H S
Published: May 2006

Experimental analysis of complex quantitative genetic traits, such as essential hypertension, should be greatly facilitated by being able to manipulate the expression of a gene in living animals without altering the nucleotide sequence, chromosomal location, or regulatory elements of the gene. To explore this possibility, we have used targeted gene disruption and duplication to generate mice that are genetically identical [(129 x C57BL6)F1] except for having one, two, or three functional copies of the gene coding for angiotensinogen. The two-copy animals have two normal copies of the angiotensinogen gene; the one-copy and three-copy animals have one normal copy with the other either disrupted or duplicated by gene targeting. The duplicated pair of genes was generated by a special form of gap-repair gene targeting that tandemly duplicates the whole of a gene together with 5' and 3' flanking regions. We find progressively and significantly higher levels of the gene product in the animals having increasing numbers of gene copies: the one-copy animals have steady-state plasma angiotensinogen levels approximately 35% of normal (P < 0.0001), and the three-copy animals have levels approximately 124% of normal (P < 0.004). Detailed information about regulatory sequences is not required for this type of experiment; nor is it necessary to have DNA clones or targeting constructs that cover the whole of the target gene. Varying gene copy numbers by targeting consequently offers a promising approach to quantitative genetics.
Functionally active targeting domain of the beta-adrenergic receptor kinase: an inhibitor of G beta gamma-mediated stimulation of type II adenylyl cyclase.
Authors: Inglese J , Luttrell L M , Iñiguez-Lluhi J A , Touhara K , Koch W J , Lefkowitz R J
Published: May 2006

The beta-adrenergic receptor kinase (beta ARK) phosphorylates its membrane-associated receptor substrates, such as the beta-adrenergic receptor, triggering events leading to receptor desensitization. beta ARK activity is markedly stimulated by the isoprenylated beta gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G beta gamma), which translocates the kinase to the plasma membrane and thereby targets it to its receptor substrate. The amino-terminal two-thirds of beta ARK1 composes the receptor recognition and catalytic domains, while the carboxyl third contains the G beta gamma binding sequences, the targeting domain. We prepared this domain as a recombinant His6 fusion protein from Escherichia coli and found that it had both independent secondary structure and functional activity. We demonstrated the inhibitory properties of this domain against G beta gamma activation of type II adenylyl cyclase both in a reconstituted system utilizing Sf9 insect cell membranes and in a permeabilized 293 human embryonic kidney cell system. Gi alpha-mediated inhibition of adenylyl cyclase was not affected. These data suggest that this His6 fusion protein derived from the carboxyl terminus of beta ARK1 provides a specific probe for defining G beta gamma-mediated processes and for studying the structural features of a G beta gamma-binding domain.
Recombination between defective tombusvirus RNAs generates functional hybrid genomes.
Authors: White K A , Morris T J
Published: May 2006

The tombusviruses represent a group of small icosahedral plant viruses that contain monopartite positive-sense RNA genomes. Tombusviruses are able to generate small replicating deletion mutants of their genomes (i.e., defective interfering RNAs) during infections via RNA recombination and/or rearrangement. To further study the process of RNA recombination and to determine whether tombusviruses were capable of trans-recombination, protoplasts were coinoculated with in vitro-generated transcripts of a nonreplicating 3'-truncated genomic RNA of cucumber necrosis tombusvirus and either replicative or replication-defective DI RNAs of tomato bushy stunt tombusvirus. After a 48-hr incubation, two dominant replicative chimeric recombinant viral RNA populations were detected that contained various large contiguous 5' segments of the cucumber necrosis tombusvirus genomic RNA fused to 3'-terminal regions of the tomato bushy stunt tombusvirus defective interfering RNA. Some of the larger chimeric recombinants formed in protoplasts were able to systemically infect plants and induce wild-type symptoms. In addition, a functional chimeric genome was generated in planta after direct coinoculation of whole plants with the defective RNA components. These results indicate that (i) RNA recombination can occur relatively efficiently in single-cell infections, (ii) trans-recombination can occur with nonreplicating viral RNA components, and (iii) functional chimeric genomes can be generated via recombination. Possible mechanisms for the formation of the recombinants are proposed, and evolutionary implications are discussed.
A faster-acting and more potent form of tissue plasminogen activator.
Authors: Keyt B A , Paoni N F , Refino C J , Berleau L , Nguyen H , Chow A , Lai J , Peña L , Pater C , Ogez J
Published: May 2006

Current treatment with tissue plasminogen activator (tPA) requires an intravenous infusion (1.5-3 h) because the clearance of tPA from the circulation is rapid (t 1/2 approximately 6 min). We have developed a tPA variant, T103N,N117Q, KHRR(296-299)AAAA (TNK-tPA) that has substantially slower in vivo clearance (1.9 vs. 16.1 ml per min per kg for tPA in rabbits) and near-normal fibrin binding and plasma clot lysis activity (87% and 82% compared with wild-type tPA). TNK-tPA exhibits 80-fold higher resistance to plasminogen activator inhibitor 1 than tPA and 14-fold enhanced relative fibrin specificity. In vitro, TNK-tPA is 10-fold more effective at conserving fibrinogen in plasma compared to tPA. Arterial venous shunt models of fibrinolysis in rabbits indicate that TNK-tPA (by bolus) induces 50% lysis in one-third the time required by tPA (by infusion). TNK-tPA is 8- and 13-fold more potent in rabbits than tPA toward whole blood clots and platelet-enriched clots, respectively. TNK-tPA conserves fibrinogen and, because of its slower clearance and normal clot lysis activity, is effective as a thrombolytic agent when given as a bolus at a relatively low dose.
Primary structure and functional expression of a guinea pig kappa opioid (dynorphin) receptor.
Authors: Xie G X , Meng F , Mansour A , Thompson R C , Hoversten M T , Goldstein A , Watson S J , Akil H
Published: May 2006

A full-length cDNA encoding the guinea pig kappa opioid (dynorphin) receptor has been isolated. The deduced protein contains 380 aa and seven hydrophobic alpha-helices characteristic of the G protein-coupled receptors. This receptor is 90% identical to the mouse and rat kappa receptors, with the greatest level of divergence in the N-terminal region. When expressed in COS-7 cells, the receptor displays high affinity and stereospecificity toward dynorphin peptides and other kappa-selective opioid ligands such as U50, 488. It does not bind the mu- and delta-selective opioid ligands. The expressed receptor is functionally coupled to G protein(s) to inhibit adenylyl cyclase and Ca2+ channels. The guinea pig kappa receptor mRNA is expressed in many brain areas, including the cerebellum, a pattern that agrees well with autoradiographic maps of classical guinea pig kappa binding sites. Species differences in the pharmacology and mRNA distribution between the cloned guinea pig and rat kappa receptors may be worthy of further examination.
Na(+)-I- symport activity is present in membrane vesicles from thyrotropin-deprived non-I(-)-transporting cultured thyroid cells.
Authors: Kaminsky S M , Levy O , Salvador C , Dai G , Carrasco N
Published: May 2006

The active accumulation of I- in the thyroid gland is mediated by the Na(+)-I- symporter and driven by the Na+ gradient generated by the Na+/K(+)-ATPase. Thyrotropin (TSH) stimulates thyroidal I- accumulation. Rat thyroid-derived FRTL-5 cells require TSH to accumulate I-. TSH withdrawal for over 7 days results in complete loss of Na(+)-I-symport activity in these cells [Weiss, S. J., Philp, N. J. and Grollman, E. F. (1984) Endocrinology 114, 1090-1098]. Surprisingly, membrane vesicles prepared from FRTL-5 cells maintained in TSH-free medium [TSH(-)cells]accumulate I-, suggesting that the absence of Na(+)-I- symport activity in TSH(-) cells cannot be due solely to a decrease in the biosynthesis of either the symporter or a putative activating factor. This finding indicates that the Na(+)-I- symporter is present, probably in an inactive state, in TSH(-) cells despite their lack of Na(+)-I- symport activity. Na(+)-I- symport activity in thyroid membrane vesicles is enhanced when conditions for vesicle preparation favor proteolysis. Subcellular fractionation studies in both TSH(+) and TSH(-) cells show that Na(+)-I- symport activity is mostly associated with fractions enriched in plasma membrane rather than in intracellular membranes, suggesting that the Na(+)-I- symporter may constitutively reside in the plasma membrane and may be activated by TSH.
Synergistic effects of bombesin and epidermal growth factor on cancers.
Authors: Liebow C , Crean D H , Lee M T , Kamer A R , Mang T S , Schally A V
Published: May 2006

Bombesin and gastrin-releasing peptide act as autocrine mitogens in various cancers. Bombesin antagonist RC-3095 inhibited growth in some cancers and slowed the progression of premalignant lesions, possibly by down-regulating epidermal growth factor (EGF) receptors. Since the EGF receptor mitogen response involves tyrosine kinase stimulation, we tested the hypotheses that bombesin stimulates, and RC-3095 inhibits, phosphorylation; EGF and bombesin promote the phosphorylation of the same substrates; and EGF and bombesin act synergistically on phosphorylation. Therefore, in vitro assays for phosphorylation were performed in the presence or absence of EGF, bombesin, RC-3095, and combinations in samples derived from tumor, tissue surrounding tumor, cell lines, and normal and transforming tissue derived from the 9,10-dimethyl-1,2-benzanthracene-induced squamous cell lesions of the hamster cheek pouch. Bombesin increased, and RC-3095 decreased, phosphorylation in these samples. In the human hepatoma sample and surrounding tissue, these ligands altered the phosphorylation of the same substrates affected by EGF. EGF and bombesin stimulated phosphorylation synergistically in the hamster samples and the hepatoma. Bombesin-induced phosphorylation was greater in tissue surrounding the hepatoma, whereas RC-3095 was more effective in inhibiting phosphorylation in the hepatoma itself. This cancer, therefore, could be endogenously stimulated by gastrin-releasing peptide. These observations support the hypothesis that bombesin stimulates growth of tissues and tumors by amplifying the phosphorylation response to EGF. The growth inhibitory response to RC-3095, or other bombesin analogues, of individual tumors may be prognosed by in vitro phosphorylation assays using the samples from the patient's tumor.
Redox modulation of the expression of bacterial genes encoding cysteine-rich proteins in plant protoplasts.
Authors: Piñeiro M , García-Olmedo F , Diaz I
Published: May 2006

Activity of neomycin phosphotransferase II (NPTII; gene, neo; five cysteines) in tobacco protoplasts transfected with fusions of the octopine TR2' or cauliflower mosaic virus 35S promoter and the neo gene, with or without a signal peptide, increased up to 8-fold in response to externally added dithiothreitol at concentrations that did not affect protoplast viability (up to 2.5 mM). Activity of phosphinothricin acetyltransferase (PAT; gene, bar; one cysteine) expressed under control of the TR1' or 35S promoter was not similarly affected, thus excluding a redox modulation of transcription as the mechanism of NPTII activation by dithiothreitol. Western-blot analyses showed an increase in the amount of protein in response to dithiothreitol, whereas neither the steady-state level of NPTII mRNA nor the specific activity of the purified enzyme was affected. The same type of modulation was observed for transiently expressed beta-glucuronidase (nine cysteines) produced from a fusion with the 35S promoter, with or without a signal peptide. Limitation of cotranslational and/or early posttranslational steps by excessively oxidizing sulfhydryl/disulfide redox potentials is postulated to explain the low net accumulation of cysteine-rich proteins of bacterial origin (i.e., NPTII and beta-glucuronidase) when expressed in plant protoplasts, and the marked increase in such proteins in response to externally added dithiothreitol.
Structure and function in rhodopsin: replacement by alanine of cysteine residues 110 and 187, components of a conserved disulfide bond in rhodopsin, affects the light-activated metarhodopsin II state.
Authors: Davidson F F , Loewen P C , Khorana H G
Published: May 2006

A disulfide bond that is evidently conserved in the guanine nucleotide-binding protein-coupled receptors is present in rhodopsin between Cys-110 and Cys-187. We have replaced these two cysteine residues by alanine residues and now report on the properties of the resulting rhodopsin mutants. The mutant protein C110A/C187A expressed in COS cells resembles wild-type rhodopsin in the ground state. It folds correctly to bind 11-cis-retinal and form the characteristic rhodopsin chromophore. It is inert to hydroxylamine in the dark, and its stability to dark thermal decay is reduced, relative to that of the wild type, by a delta delta G not equal to of only -2.9 kcal/mol. Further, the affinities of the mutant and wild-type rhodopsins to the antirhodopsin antibody rho4D2 are similar, both in the dark and in light. However, the metarhodopsin II (MII) and MIII photointermediates of the mutant are less stable than those formed by the wild-type rhodopsin. Although the initial rates of transducin activation are the same for both mutant and wild-type MII intermediates at 4 degrees C, at 15 degrees C the MII photointermediate in the mutant decays more than 20 times faster than in wild type. We conclude that the disulfide bond between Cys-110 and Cys-187 is a key component in determining the stability of the MII structure and its coupling to transducin activation.
Negative selection of CD4+ CD8+ thymocytes by T-cell receptor peptide antagonists.
Authors: Page D M , Alexander J , Snoke K , Appella E , Sette A , Hedrick S M , Grey H M
Published: May 2006

Antigen-induced activation of T cells can be specifically inhibited by antigen analogs that have been termed T-cell receptor peptide antagonists. These antagonists appear to act by inducing the formation of nonstimulatory or partially stimulatory complexes between T-cell receptors and the major histocompatibility complex molecules presenting the peptides. Herein, we have investigated the effect of T-cell receptor peptide antagonists on thymocyte negative selection. First, peptide antagonists were identified for the cytochrome c-specific T-cell clone AD10. These peptides were then tested for their ability to induce negative selection in an in vitro model system using thymocytes from mice transgenic for the AD10 T-cell receptor. Though unable to induce mature T-cell activation, the T-cell receptor peptide antagonists induced deletion of CD4+ CD8+ thymocytes. These results suggest that negative selection of CD4+ CD8+ thymocytes can be induced by T-cell receptor interactions of a lower affinity than those required for mature T-cell activation.
Expression of an active glycosylated human gamma-glutamyl transpeptidase mutant that lacks a membrane anchor domain.
Authors: Ikeda Y , Fujii J , Taniguchi N , Meister A
Published: May 2006

A mutant of human gamma-glutamyl transpeptidase (EC 2.3.2.2, a membrane-bound enzyme of importance in glutathione metabolism) that differs from the wild type by deletion of the putative signal peptide/anchor domain (amino acid residues 1-27) was expressed in insect cells using a baculovirus system. In contrast to the wild-type enzyme--which, as expected, was mainly cell-associated--the mutant enzyme was secreted into the medium. The mutant and wild-type enzymes were purified and found to exhibit virtually identical catalytic properties. The mutant enzyme was glycosylated and processed into two subunits, as found for the wild-type enzyme. Brefeldin A inhibited secretion of the mutant enzyme and led to its accumulation in cells. The findings indicate that gamma-glutamyl transpeptidase can be targeted to the endoplasmic reticulum in a manner that does not involve function of an amino-terminal "signal/anchor" domain and that this domain is involved primarily in a membrane anchoring function. Another region of the enzyme may function as a signal domain.
Comparative genomic analysis of tumors: detection of DNA losses and amplification.
Authors: Lisitsyn N A , Lisitsina N M , Dalbagni G , Barker P , Sanchez C A , Gnarra J , Linehan W M , Reid B J , Wigler M H
Published: May 2006

We demonstrate the use of representational difference analysis for cloning probes that detect DNA loss and amplification in tumors. Using DNA isolated from human tumor cell lines to drive hybridization against matched normal DNA, we were able to identify six genomic regions that are homozygously deleted in cultured cancer cells. When this method was applied in the reverse way, using normal DNA to drive hybridization against tumor cell DNA, we readily isolated probes detecting amplification. Representational difference analysis was also performed on DNAs derived from tumor biopsies, and we thereby discovered a probe detecting very frequent homozygous loss in colon cancer cell lines and located on chromosome 3p.
Rapid induction of tumor necrosis factor alpha in the cerebrospinal fluid after intracerebroventricular injection of lipopolysaccharide revealed by a sensitive capture immuno-PCR assay.
Authors: Sanna P P , Weiss F , Samson M E , Bloom F E , Pich E M
Published: May 2006

Tumor necrosis factor alpha (TNF-alpha) is an important mediator in many pathophysiologic processes, both in the central nervous system (CNS) and in the periphery. For this study, we have designed a very sensitive immuno-PCR detection system to investigate the time course of TNF-alpha induction in the rat cerebrospinal fluid after intracerebroventricular administration of bacterial lipopolysaccharide (LPS). Immuno-PCR combines antibody specificity with PCR signal amplification and provides a sensitivity in the picomolar range. The enhanced sensitivity of this assay allowed the detection of TNF-alpha in the cerebrospinal fluid as early as 15 min after intracerebroventricular administration of LPS. The present results suggest that the ventricular compartment of the CNS, although confined within the blood-brain barrier, is highly responsive to proinflammatory stimuli such as LPS administration. Insight into the molecular mechanisms underlying this compartmentalization could be key to the pathology and treatment of many CNS diseases, especially the meningitides.