Green fluorescent protein as a probe of rotational mobility within bacteriophage T4.

Green fluorescent protein (GFP) was targeted into bacteriophage T4 heads and proheads as a probe of the internal environment. Targeting was accomplished with internal protein III (IPIII) fusion proteins or capsid targeting sequence (CTS)-tagged proteins, where CTS is the 10-amino acid residue CTS of IPIII. Recombinant phage T4[CTS/IPIII/GFP], T4[CTS/IPIII(T)GFP], and T4[CTS/GFP] packaged GFP fusion proteins and processed them at cleavage sites designated /. Steady-state and time-resolved fluorescence measurements suggest that packaged GFP is concentrated to a high density, that fusion protein IPIII(T)GFP occurs in a tightly clustered arrangement, and that the internal milieu of the phage head reduces rotational mobility of GFP. Phage, but not proheads, packaged with fusion protein IPIII(T)GFP gave an unexpectedly lower anisotropy than phage and proheads packaged with GFP, which suggests IPIII(T)GFP is bound to DNA in a manner that causes close associations between GFP molecules resulting in homotransfer between fluorophores within packaged phage. Targeting of reporter proteins into active virions is a promising approach for determining the structure of the condensed DNA, and properties of encapsidated viral enzymes.

GFP:HIV-1 protease production and packaging with a T4 phage expression-packaging processing system.

A bacteriophage T4-derived protein expression, packaging and processing system was used to create recombinant phage that encode, produce and package a protein composed of human HIV-1 protease fused to green fluorescent protein (GFP). The fusion protein is targeted within the phage capsid by an N-terminal capsid targeting sequence (CTS), which is cleaved through proteolysis by the viral scaffold protease P21. The fusion protein is designated CTS [symbol see text] GFP:PR. The [symbol see text] symbol indicates the linkage peptide sequence leu(ile)-N-glu that is cleaved by the T4 head morphogenetic proteinase gp21 during head maturation. The fusion protein is fluorescent and has protease activity as detected by the appearance of the expected substrate cleavage product on a Western blot. CTS [symbol see text] GFP:PR packaging occurs at about 200 molecules per phage particle. The CTS [symbol see text] GFP:PR fusion protein, when protected within the phage capsid, has been maintained stably for over 16 months at 4 degrees C. Production and storage of fusion protein within the phage circumvents problems of toxicity and solubility encountered with E. coli expression systems. Because recombinant phage inhibit host proteolytic enzymes, foreign proteins are stabilized. This phage system packages and processes the fusion protein by means of the CTS. Proteins can be purified from the phage to give high yields of soluble, proteolytically processed protein. The T4 phage packaging system provides a novel means of identification, purification and long-term storage of toxic proteins whose folding and DNA-directed activities can be studied readily in vivo.

Activity of foreign proteins targeted within the bacteriophage T4 head and prohead: implications for packaged DNA structure.

The phage-derived expression, packaging, and processing (PEPP) system was used to target foreign proteins into the bacteriophage capsid to probe the intracapsid environment and the structure of packaged DNA. Small proteins with minimal requirements for activity were selected, staphylococcal nuclease (SN) and green fluorescent protein (GFP). These proteins were targeted into the T4 head by means of IPIII (internal protein III) fusions or CTS (capsid targeting sequence) fusions. Additional evidence is provided that foreign proteins are targeted into T4 by the N-terminal ten amino acid residue consensus CTS of IPIII identified in previous work. Fusion proteins were produced within host bacteria by expression from plasmids or by produc tion from recombinant phage carrying the fusion genes. Packaged fusion proteins CTS IPIII SN, CTS IPIII TSN, CTS IPIII GFP, CTS IPIII TGFP, and CTS GFP, where [symbol: see text] indicates a linkage peptide sequence Leu(Ile)-N-Glu cleaved by the T4 head morphogenetic proteinase gp21 during head maturation, are observed to exhibit intracapsid activity. SN activity within the head is demonstrated by loss of phage viability and by digested genomic DNA patterns visualized by gel electrophoresis when viable phage are incubated in Ca2+. Green fluorescent phage result immediately after packaging GFP produced at 30 degreesC and below, and continue to give green fluorescence under 470 nm light after CsCl purification. Non-fluorescent GFP-fusions are produced in bacteria at 37 degreesC, and phage packaged with these proteins achieve a fluorescent state after incubation for several months at 4 degreesC. GFP-packaged phage and proheads analyzed by fluorescence spectroscopy show that the mature head and the DNA-empty prohead package identical numbers of GFP-fusion proteins. Encapsidated GFP and SN can be injected into bacteria and rapidly exhibit intracellular activity. In vivo SN digestion of encapsidated DNA gives an intriguing pattern of DNA fragments by gel analysis, predominantly a repeat pattern of 160 bp multiples, reminiscent of a nucleosome digestion ladder, This quasi-limit DNA digestion pattern, reached >100-fold more slowly than the loss of titer, is invariant over a range

Primary adrenal lymphoma: gallium scintigraphy and correlative imaging.

Primary adrenal lymphoma is a rare entity, with only 16 cases reported in the last 40 yr. Although 67Ga scintigraphy has been extensively used to evaluate patients with other types of lymphomas, there are no reports of its use in patients with this disease entity. A man with primary adrenal lymphoma and no evidence of extraadrenal spread who was evaluated from presentation to remission with gallium scintigraphy and CT is presented. Gallium scintigraphy was valuable in assessing response to therapy.

Capsid targeting sequence targets foreign proteins into bacteriophage T4 and permits proteolytic processing.

A membrane-independent morphogenetic viral signal peptide is identified within bacteriophage T4 internal protein III (IPIII). Utilizing a phagederived expression-packaging-processing system, which packages foreign proteins fused with IPIII into the phage capsid, a synthetic cleavage site introduced at the C terminus of IPIII, is demonstrated to be functional and permits processing of fusion proteins. IPIII, which possesses a native P21 cleavage site at its N terminus, is altered to possess a second P21 cleavage site at its C terminus where cleavage occurs by means of the scaffold proteinase P21 within the capsid. The altered IPIII was inserted into an expression vector to permit the creation of fusion proteins with staphylococcal nuclease, EcoRI endonuclease, beta-globin, and luciferase. Western immunoblot analysis of packaged T4eG326 indicates that the IPIII:fusion-proteins are packaged into phage and proteolytically processed, thus the synthetic P21 cleavage site positioned at the C terminus of IPIII is demonstrated to be functional, and 20 to 200 protein molecules are packaged per capsid. Truncation experiments identified the minimal portion of IPIII required to achieve targeting into the phage capsid as a ten amino acid residue from the N terminus, which includes the N-terminal methionine residue and the proteinase P21 cleavage site, designated the CTS (capsid targeting sequence). The addition of the CTS to a fragment of luciferase permits the protein to be packaged and processed, which demonstrates that the CTS is by itself sufficient to target foreign protein to the capsid. The imputed dual function of the CTS is supported by site-directed PCR mutagenesis, which reveals two functionally separate domains of the CTS for targeting and processing. The CTS appears to function in a core-related targeting mechanism that directs a polymorphic set of proteins into the T-even capsid or scaffold. Although structure formation is often assumed to involve extended protein interfaces, the analysis shows that a limited but specific sequence, the CTS, drives the interaction required to achieve targeting.

Enzymatic digestion of the urethra after pancreatic transplantation: a case report.

The most common method of exocrine drainage in pancreatic transplantation is urinary drainage. With the shift from enteric drainage and duct occlusion techniques, there has been a concomitant shift from intraabdominal to urological complications. We present a case in which a simultaneous pancreas-kidney (SPK) transplant recipient developed urethral disruption and associated bladder outlet obstruction 11 months following surgery.

Protection from proteolysis using a T4::T7-RNAP phage expression-packaging-processing system.

DNA coding for bacteriophage T7 RNA polymerase (T7-RNAP) was inserted into a positive selection-vector form of the T4 genome, placing it under the control of bacteriophage T4 ipIII promoters. The recombinant T4::T7-RNAP fusion phage retained infectivity and produced T7-RNAP in infected cells. Fusion genes were constructed by insertion into a plasmid containing an iPIII (encoding internal protein III) target portion and a bacteriophage T7 promoter region. When Escherichia coli cells containing the plasmid were infected with the T4::T7-RNAP re-phage, the bacteria produced fusion protein at high levels. The newly synthesized T4::T7-RNAP re-phage progeny package and process the fusion protein into the phage capsid during head morphogenesis. In this paper, we demonstrate that truncated T4 internal protein IPIII, human IPIII::beta Glo (beta-globin) fusion protein, E. coli IPIII::beta Glo::beta Gal (beta-galactosidase) triple-fusion protein and IPIII::V3 fusion protein (human immunodeficiency virus envelope protein gp120 V3 region) are expressed at high levels by T4::T7-RNAP induction. With IPIII::beta Glo, expression-packaging-processing (EPP) occurs simultaneously with T4::T7-RNAP re-phage infection. We also demonstrate that T4::T7-RNAP re-phage stabilize unstable proteins such as the X90 fragment of beta Gal, thought to be degraded by the lon protease. An unstable 20-kDa fragment of the large subunit of human cytochrome b558, an integral membrane protein in phagocytes, is subject to proteolytic degradation even when produced in the lon-deficient BL21 strain. However, upon induction with T4::T7-RNAP re-phage, the 20-kDa protein is produced intact.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium signalling mechanisms in endoplasmic reticulum activated by inositol 1,4,5-trisphosphate and GTP.

Ca2+ signals are known to mediate an array of cellular functions including secretion, contraction, and conductivity changes. In spite of the obvious role of Ca2+ in signalling, the control of Ca2+ within cells is known to be a complex phenomenon involving a number of distinct active and passive transport systems functioning within different organelles. Inositol 1,4,5-trisphosphate (IP3) is now established as a central mediator of Ca2+ mobilization, and the endoplasmic reticulum (ER) has been considered to be the site of action of IP3. However, this role has been ascribed almost by default to the ER, based on the knowledge that IP3 functions to release Ca2+ from an intracellular, nonmitochondrial, Ca2+-pumping organelle. Our interest has been to ascertain by what mechanism IP3 activates Ca2+ movements, at what intracellular locations it functions, and how the size and replenishment of the IP3-sensitive Ca2+ pool occurs. During the course of such studies, another mechanism inducing profound movements of Ca2+ within cells was identified. This process is activated by a highly sensitive and specific guanine nucleotide regulatory mechanism, which, while inducing fluxes of Ca2+ that resemble the action of IP3 under certain conditions, has now been determined to involve a quite distinct mechanism. The characteristics of this mechanism are described below. Although involving a very different Ca2+ translocation process to that activated by IP3, several important conclusions have been drawn on the relationship between IP3- and GTP-activated Ca2+ movements leading us to believe that the latter may have a regulatory role in controlling the size and/or entry of Ca2+ into the IP3-sensitive Ca2+ pool.

GTP-activated communication between distinct inositol 1,4,5-trisphosphate-sensitive and -insensitive calcium pools.

Inositol 1,4,5-trisphosphate (InsP3) is an established mediator of intracellular Ca2+ signals but little is known of the nature and organization of Ca2+ regulatory organelles responsive to InsP3. Here we derive new information from the study of Ca2+ movements induced both by InsP3 and a specific GTP-activated Ca2+ translocation process. The latter mechanism is clearly distinct from that activated by InsP3 and may involve the translocation of Ca2+ between compartments without its release into the cytosol. This idea is supported by the fact that GTP activates Ca2+ movement into the InsP3-releasable pool. In the light of this evidence we postulated that there are two intracellular Ca2+ pools distinguishable by InsP3-sensitivity and oxalate-permeability, and that movement between them is activated by GTP. We report here direct evidence for the existence and separation of two distinct Ca2+-pumping compartments with properties coinciding with those predicted. Of these, the InsP3-sensitive Ca2+ pool is identified within a purified rough endoplasmic reticulum fraction, an observation consistent with recent InsP3 receptor-localization studies. Ca2+ translocation between pools may reflect function of a class of small GTP-binding proteins known to mediate interorganelle transfer in eukaryotic cells.

Intracellular calcium translocation: mechanism of activation by guanine nucleotides and inositol phosphates.

The movements of Ca2+ within cells in response to external stimuli are complex. Internal Ca2+ release activated by inositol 1,4,5-trisphosphate (InsP3) is now widely established. However, the mechanism of InsP3-induced Ca2+ release, the identity and control of the InsP3-sensitive Ca2+ pool and its relationship to other internal and external Ca2+ pools all remain uncertain. We have characterized a highly sensitive and specific guanine nucleotide-regulatory mechanism that induces rapid and profound movements of intracellular Ca2+ via a mechanism distinct from that activated by InsP3. Using permeabilized neural or smooth muscle cells, application of submicromolar concentrations of GTP induces rapid release of Ca2+ from a compartment that contains within it the InsP3-releasable Ca2+ pool. Although of similar GTP-sensitivity as G-protein-activated events, the apparent dependence on GTP hydrolysis and blockade by GTP gamma S suggest a mechanism distinct from those mediated by known G-proteins. Recent experiments in the presence of oxalate reveal rapid and profound GTP-activated uptake of Ca2+ via a mechanism with identical nucleotide sensitivity and specificity to GTP-induced Ca2+ release. These results were interpreted to suggest that GTP induces a transmembrane conveyance of Ca2+ between different compartments distinguished by oxalate permeability; GTP-induced release probably occurs via a similar mechanism except involving transfer between closed compartments and nonclosed membranes (perhaps the plasma membrane). Recently, it has been revealed that GTP activates a translocation of Ca2+ into the Ca2+ pool from which InsP3 induces release. This is an important observation suggesting that the GTP-activated Ca2+ translocation process may control entry into and hence the size of the InsP3-releasable Ca2+ pool. Indeed, it is possible that GTP-induced Ca2+ release observed in permeabilized cells reflects a reversal of the pathway that functions in intact cells to permit external Ca2+ entry into the InsP3-releasable pool. This type of process could mediate the longer-term secretory or excitatory responses to external receptors which are known to be dependent on external Ca2+.

Competitive, reversible, and potent antagonism of inositol 1,4,5-trisphosphate-activated calcium release by heparin.

The action of inositol 1,4,5-trisphosphate (InsP3) in releasing intracellular Ca2+ is shown to be competitively and potently antagonized by the glycosaminoglycan, heparin. Using either permeabilized cells of the DDT1MF-2 smooth muscle cell line, or an isolated microsomal membrane fraction derived from intact cells, heparin (4-6 kDa) at 10 micrograms/ml was observed to completely block the action of InsP3 in releasing Ca2+ accumulated via the ATP-dependent Ca2+ pump. In permeabilized cells, heparin had no effect on Ca2+ pump activity or on passive Ca2+ fluxes contributing to equilibrium Ca2+ accumulation. Heparin up to 100 micrograms/ml had no effect on the GTP-activated Ca2+ translocation process previously characterized in this cell line. Half-maximal inhibition of Ca2+ release activated by 10 microM InsP3 occurred with heparin at approximately 0.6 and 0.2 microgram/ml in permeabilized cells and isolated microsomes, respectively. Using microsomes, InsP3 dose-response curves in the presence and absence of 0.2 microgram/ml heparin (approximately 40 nM) revealed a 10-fold increase in apparent Km for InsP3 (0.31 microM in the absence of heparin) with no change in Vmax, indicating a competitive action of heparin. The results revealed a very high apparent affinity of heparin for the InsP3 active site, with a calculated Ki value of 2.7 nM. Heparin was shown to rapidly (within 20 s) reverse prior full activation of InsP3-mediated Ca2+ release returning the Ca2+ equilibrium back to that observed without InsP3. This reversal occurs even after prolonged (6 min) InsP3 activation. These results indicate a specific, high affinity, and competitive antagonism of the InsP3 active site by heparin. The rapidly induced reversal of InsP3-activated Ca2+ release by heparin strongly suggests that InsP3 directly activates a channel which remains open only while InsP3 is associated and closes immediately upon InsP3 dissociation.

Calcium entry into the inositol 1,4,5-trisphosphate-releasable calcium pool is mediated by a GTP-regulatory mechanism.

Intracellular Ca2+ release activated by inositol 1,4,5-trisphosphate (InsP3) plays a pivotal role in Ca2+ signaling in cells. A controlling mechanism for InsP3-induced Ca2+ movements is suggested by results showing that the InsP3-releasable Ca2+ pool is directly modified by a specific and sensitive GTP-regulated Ca2+-translocating process. By using saponin-permeabilized N1E-115 neuroblastoma cells or DDT1MF-2 smooth muscle-derived cells, InsP3 releases 30-50% of Ca2+ accumulated through intracellular high-affinity ATP-dependent Ca2+-pumping activity. Oxalate-promoted Ca2+ uptake is reversed by InsP3, indicating oxalate permeability of the InsP3-releasable pool, which is consistent with this compartment being the endoplasmic reticulum. GTP (10 microM) activates release of 50-70% of accumulated Ca2+ from cells. In the presence of 5-10 mM oxalate, GTP induces a biphasic Ca2+ flux response; initially (1-2 min) GTP induces rapid Ca2+ release followed thereafter by a profound increase in Ca2+ uptake. Thus, GTP-activated Ca2+ influx and efflux compete for Ca2+ access to the oxalate-permeable Ca2+ pool. The nonadditive effects of InsP3 and GTP suggest that InsP3 releases Ca2+ from a subcompartment of the GTP-releasable pool. Most significantly, InsP3 is observed to block the GTP-activated uptake phase in the presence of oxalate, indicating that GTP induces Ca2+ entry into the pool from which InsP3 activates release. Hence, the results provide direct evidence that loading of Ca2+ into the InsP3-sensitive Ca2+ pool is controlled by a GTP-regulated Ca2+-translocating mechanism. Such a process could be significant in regulating the extent and duration of the InsP3-induced Ca2+ signal, a crucial step in the inositol phospholipid signaling pathway.

Intracellular calcium uptake activated by GTP. Evidence for a possible guanine nucleotide-induced transmembrane conveyance of intracellular calcium.

The GTP-activated Ca2+ release process we recently described (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464) was revealed in the preceding report to operate via a mechanism likely to be induced by close membrane association but which appears not to involve membrane fusion (Chueh, S. H., Mullaney, J. M., Ghosh, T. K., Zachary, A. L., and Gill, D. L. (1987) J. Biol. Chem. 262, 13857-13864). To determine more about the GTP-activated Ca2+ translocation process, effects of GTP on cells loaded with Ca-oxalate were investigated. Using permeabilized cells of both the N1E-115 neuroblastoma and DDT1MF-2 smooth muscle cell lines, 10 microM GTP activates a profound uptake of Ca2+ in the presence of oxalate, as opposed to release observed without oxalate. GTP stimulation of Ca2+ uptake was observed at oxalate concentrations (2 mM) only slightly augmenting Ca2+ uptake without GTP; with 8 mM oxalate (which alone induces linear Ca2+ accumulation) GTP still increases the rate of uptake. GTP-activated uptake in the presence of oxalate is completely reversed by 1 mM vanadate. 3% polyethylene glycol enhances the effect of GTP although GTP-activated uptake is still observed without polyethylene glycol. The Km for GTP for activation of Ca2+ uptake is 0.9 microM. Uptake is not activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp); however, GTP gamma S (but not GppNHp) completely blocks the action of GTP. GDP gives a delayed uptake response which is blocked by ADP, indicating its action arises from conversion to GTP. In the presence of ADP, GDP blocks the action of GTP; guanosine 5'-O-(2-thio)diphosphate, which does not activate uptake, also blocks the action of GTP. These data reveal almost exact correlation between parameters affecting GTP-activated uptake and release, strongly suggesting the same process mediates both events. To explain the opposite effects of GTP in the absence and presence of oxalate, it is proposed that GTP activates a transmembrane conveyance of Ca2+ between oxalate-permeable and -impermeable compartments.

GTP- and inositol 1,4,5-trisphosphate-activated intracellular calcium movements in neuronal and smooth muscle cell lines.

Recent evidence has revealed that a highly sensitive and specific guanine nucleotide regulatory process controls intracellular Ca2+ release within N1E-115 neuroblastoma cells (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464). The present report documents GTP-induced Ca2+ release within quite distinct cell types, including the DDT1MF-2 smooth muscle cell line. GTP-induced Ca2+ release has similar GTP sensitivity and specificity among cells and rapidly mobilizes up to 70% of Ca2+ specifically accumulated within a nonmitochondrial Ca2+-pumping organelle within permeabilized DDT2MF-2 cells. Maximal GTP-induced release of Ca2+ is observed to be greater than inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release (the latter being approximately 30% of total releasable Ca2+). After maximal IP3-induced release, further IP3 addition is ineffective, whereas subsequent addition of GTP further releases Ca2+ to equal exactly the extent of Ca2+ release observed by addition of GTP in the absence of IP3. This suggests that IP3 releases Ca2+ from the same pool as GTP, whereas GTP also releases from an additional pool. The effects of GTP appear to be reversible since simple washing of GTP-treated cells restores their previous Ca2+ uptake properties. Electron microscopic analysis of GTP-treated membrane vesicles reveals their morphology to be unchanged, whereas treatment of vesicles with 3% polyethylene glycol, known to enhance GTP-mediated Ca2+ release, clearly induces close coalescence of membranes. In the presence of 4 mM oxalate, GTP induces a rapid and profound uptake, as opposed to release, of Ca2+. The findings suggest that GTP-activated Ca2+ movement is a widespread phenomenon among cells, which can function on the same Ca2+ pool mobilized by IP3, and although activating Ca2+ movement by a mechanism distinct from IP3, does so via a process that does not appear to involve fusion between membranes.