Inositol 1,4,5-trisphosphate receptors (InsP3R) in Xenopus laevis oocytes were localized and their regulation by Ca2+ was investigated. Antibodies raised against the C-terminal region of the mouse cerebellar InsP3R (cAb) cross-reacted with a 255 kD protein in Western blots of Xenopus microsomal membranes. Immunolocalization of this protein in cryosections of oocytes revealed diffuse staining of the cytoplasm, intense staining of the sub-plasma membrane region of the animal hemisphere, and punctate staining in association with the germinal vesicle. In the presence of 40 microM free Ca2+, isolated oocyte membranes exhibited a high affinity binding site for Ins 1,4,5-P3 (KD = 5nM) and a binding capacity of 450 fmol/mg protein. The specific binding capacity of oocyte membranes for [3H]-Ins 1,4,5-P3 increased as the level of free Ca2+ present in binding assays was raised from < 0.1 nM to 4.0 microM, with an apparent EC50 of 60 nM. Increasing the concentration of free Ba2+ failed to facilitate [3H]-Ins1,4,5-P3 binding. Other inositol phosphates competed for Ins1,4,5-P3 binding sites with approximate IC50 values of: Ins1,3,4,5-P4 = 79 nM, Ins2,4,5-P3 = 455 nM and L-Ins1,4,5-P3 = 20 microM. In addition, 150 micrograms/ml (approximately 12 microM) heparin displaced 50% of bound [3H]-Ins1,4,5-P3, whereas caffeine (10 mM) had little effect. Functional reconstitution of solubilized InsP3Rs into lipid bilayers revealed that Ca2+ was a necessary co-agonist for activation of the InsP3R. When InsP3 (5 microM) and Ca2+ (5 microM) were applied together, conductance steps were observed. InsP3 or Ca2+ alone had little effect. These results suggest that the subcellular organization of InsP3Rs and the facilitation of InsP3 binding and channel opening by Ca2+ contribute to the Ins1,4,5-P3-mediated Ca2+ spikes, waves, and oscillations observed in Xenopus oocytes.

Membranes of HL-60 cells were shown to possess saturable binding sites for [3H]inositol(1,3,4,5)tetrakisphosphate, with nanomolar affinity (KD = 90 nM) and a density of 250 fmol/mg protein. The specificity of the binding sites for Ins(1,3,4,5)P4 was assessed by competition studies utilising a variety of inositol polyphosphates; results indicated that both the presence and the correct grouping of the phosphates were important for high affinity recognition. The apparent affinity of the binding sites for Ins(1,3,4,5)P4 was over 200-fold greater than for Ins(1,4,5)P3. The possibility is discussed that this binding site represents the receptor which mediates the action of Ins(1,3,4,5)P4 as a putative intracellular second messenger.

The response of cells to many external stimuli requires a decoding process at the membrane to transduce information into intracellular messengers. A major decoding mechanism employed by a variety of hormones, neurotransmitters and growth factors depends on the hydrolysis of a unique inositol lipid to generate two key second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Here I examine the second messenger function of Ins(1,4,5)P3 in controlling the mobilization of calcium. We know most about how this messenger releases calcium from internal reservoirs but less is known concerning the entry of external calcium. One interesting possibility is that Ins(1,4,5)P3 might function in conjunction with its metabolic product Ins(1,3,4,5)P4 to control calcium entry through a mechanism employing a region of the endoplasmic reticulum as a halfway house during the transfer of calcium from outside the cell into the cytoplasm. The endoplasmic reticulum interposed between the plasma membrane and the cytosol may function as a capacitor to insure against the cell being flooded with external calcium. When stimulated, cells often display remarkably uniform oscillations in intracellular calcium. At least two oscillatory patterns have been recognized suggesting the existence of separate mechanisms both of which may depend upon Ins(1,4,5)P3. In one mechanism, oscillations may be driven by periodic pulses of Ins(1,4,5)P3 produced by receptors under negative feedback control of protein kinase C. The other oscillatory mechanism may depend upon Ins(1,4,5)P3 unmasking a process of calcium-induced calcium release from the endoplasmic reticulum. The function of these calcium oscillations is still unknown. This Ins(1,4,5)P3/calcium signalling system is put to many uses during the life history of a cell.(ABSTRACT TRUNCATED AT 250 WORDS)

RNA polymerase (RNAP) isolated from Staphylococcus aureus is deficient in sigma factor and is poorly active in transcription assays. Based on amino acid sequence homology of the Bacillus subtilis vegetative sigma factor sigmaA and the predicted product of the chromosomally located plaC gene of S. aureus, it was hypothesized that plaC could encode the vegetative sigma factor. We cloned plaC under a T7 promoter and overexpressed it in Escherichia coli strain BL21(DE3)pLysE. The overproduced protein, present in inclusion bodies, was solubilized with guanidine hydrochloride, renatured, and purified by DEAE-Sephacel and Sephadex G-75 chromatography. The purified protein, designated sigmaSA, cross-reacted with the B. subtilis anti-sigmaA antibody. E. coli core RNAP, reconstituted with sigmaSA, initiated promoter-specific transcription from the S. aureus promoters hla, sea, and sec and from the E. coli promoters rpoH P1, rpoH P4, and ColE1 RNA-1, which are recognized by the E. coli sigma70. sigmaSA, when added to the purified RNAP from S. aureus, stimulated transcriptional activity of the RNAP up to 72-fold. As determined by primer extension studies, the 5'-ends of the sigmaSA-initiated mRNAs synthesized in vitro from the agr P2 and sea promoters are in general agreement with the 5'-ends of the cellular RNAs. Disruption of the plaC gene on the S. aureus chromosome was lethal. We conclude that plaC encodes the primary sigma factor in S. aureus.

This paper is a comprehensive, critical review of the levels, behaviour and processes affecting polychlorinated dibenzo-p-dioxins and -furans (PCDD/Fs) in air and deposition. Aspects of sampling, analysis and quality assurance/control are discussed initially, before a review of the PCDD/F concentrations in ambient air is presented. The general trend in sigma P4-8 CDD/F (and sigma TEQ) is: remote sites < 0.5 pg/m3 (sigma TEQ < 10 fg/m3); rural sites approximately 0.5-4 pg/m3 (sigma TEQ approximately 20-50 fg/m3); and urban/industrial sites approximately 10-100 pg/m3 (sigma TEQ approximately 100-400 fg/m3). The commonly held view that a consistent mixture of PCDD/Fs in air exists is evaluated and questioned. Issues of seasonality and short-term changes in air concentrations are also critically discussed, with respect to the possibility of seasonal emission sources to air and seasonally dependent loss processes. Data on the gas-particle partitioning of PCDD/Fs in air are reviewed; the limited database to date is believed to provide evidence for an exchangeable transfer of PCDD/Fs between these two phases. The potential importance of photolytic and radical reaction degradation processes and wet/dry deposition processes in modifying the mixture of PCDD/Fs in air is discussed. Some homologue/congener specific 'weathering' of the mixture of PCDD/Fs emitted to the atmosphere clearly occurs, but in general PCDD/Fs have 'long' atmospheric residence times, rendering them subject to long-range atmospheric transport. Data are reviewed which relate the mixture of PCDD/Fs in air to that in deposition; this leads to the conclusion that different homologue groups (which are partitioned differently between the gas and particulate phase) are transferred to the earth's surface with broadly similar efficiencies.

Various ovarian cell types including granulosa cells and ovarian surface epithelial cells express the progesterone (P4) binding protein, progesterone receptor membrane component-1 (PGRMC1). PGRMC1 is also expressed in ovarian tumors. PGRMC1 plays an essential role in promoting the survival of both normal and cancerous ovarian cell in vitro. Given the clinical significance of factors that regulate the viability of ovarian cancer, this review will focus on the role of PGRMC1 in ovarian cancer, while drawing insights into the mechanism of PGRMC1's action from cell lines derived from healthy ovaries as well as ovarian tumors. Studies using PGRMC1siRNA demonstrated that P4's ability to inhibit ovarian cells from undergoing apoptosis in vitro is dependent on PGRMC1. To confirm the importance of PGRMC1, the ability of PGRMC1-deplete ovarian cancer cell lines to form tumors in intact nude mice was assessed. Compared to PGRMC1-expressing ovarian cancer cells, PGRMC1-deplete ovarian cancer cells formed tumors in fewer mice (80% compared to 100% for controls). Moreover, the number of tumors derived from PGRMC1-deplete ovarian cancer cells was 50% of that observed in controls. Finally, the tumors that formed from PGRMC1-deplete ovarian cancer cells were about a fourth the size of tumors derived from ovarian cancer cells with normal levels of PGRMC1. One reason for PGRMC1-deplete tumors being smaller is that they had a poorly developed microvasculature system. How PGRMC1 regulates cell viability and in turn tumor growth is not known but part of the mechanism likely involves the regulation of genes that promote cell survival and inhibit apoptosis.

Mutations in HIV-1 protease (PR) that produce resistance to antiviral PR inhibitors are a major problem in AIDS therapy. The mutation F53L arising from antiretroviral therapy was introduced into the flexible flap region of the wild-type PR to study its effect and potential role in developing drug resistance. Compared to wild-type PR, PR(F53L) showed lower (15%) catalytic efficiency, 20-fold weaker inhibition by the clinical drug indinavir, and reduced dimer stability, while the inhibition constants of two peptide analog inhibitors were slightly lower than those for PR. The crystal structure of PR(F53L) was determined in the unliganded form at 1.35 Angstrom resolution in space group P4(1)2(1)2. The tips of the flaps in PR(F53L) had a wider separation than in unliganded wild-type PR, probably due to the absence of hydrophobic interactions of the side-chains of Phe53 and Ile50'. The changes in interactions between the flaps agreed with the reduced stability of PR(F53L) relative to wild-type PR. The altered flap interactions in the unliganded form of PR(F53L) suggest a distinct mechanism for drug resistance, which has not been observed in other common drug-resistant mutants.

We have identified, isolated, and characterized a second inositol polyphosphate-5-phosphatase enzyme from the soluble fraction of human platelets. The enzyme hydrolyzes inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) to inositol 1,4-bisphosphate (Ins(1,4)P2) with an apparent Km of 24 microM and a Vmax of 25 mumol of Ins(1,4,5)P3 hydrolyzed/min/mg of protein. The enzyme hydrolyzes inositol (1,3,4,5)-tetrakisphosphate (Ins(1,3,4,5)P4) at a rate of 1.3 mumol of Ins(1,3,4,5)P4 hydrolyzed/min/mg of protein with an apparent Km of 7.5 microM. The enzyme also hydrolyzes inositol 1,2-cyclic 4,5-trisphosphate (cIns(1:2,4,5)P3) and Ins(4,5)P2. We purified this enzyme 2,200-fold from human platelets. The enzyme has a molecular mass of 75,000 as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration chromatography. The enzyme requires magnesium ions for activity and is not inhibited by calcium ions. The 75-kDa inositol polyphosphate-5-phosphatase enzyme differs from the previously identified platelet inositol polyphosphate-5-phosphatase as follows: molecular size (75 kDa versus 45 kDa), affinity for Ins(1,3,4,5)P4 (Km 7.5 microM versus 0.5 microM), Km for Ins(1,4,5)P3 (24 microM versus 7.5 microM), regulation by protein kinase C, wherein the 45-kDa enzyme is phosphorylated and activated while the 75-kDa enzyme is not. The 75-kDa enzyme is inhibited by lower concentrations of phosphate (IC50 2 mM versus 16 mM for the 45-kDa enzyme) and is less inhibited by Ins(1,4)P2 than is the 45-kDa enzyme. The levels of inositol phosphates that act in calcium signalling are likely to be regulated by the interplay of these two enzymes both found in the same cell.

We have solved the crystal structure of the MHCII molecule, IA(b), containing an antigenic variant of the major IA(b)-binding peptide derived from the MHCII IEalpha chain. The four MHC pockets at p1, p4, p6, and p9 that usually bind peptide side chains are largely empty because of alanines in the peptide at these positions. The complex is nevertheless very stable, apparently because of unique alternate interactions between the IA(b) and peptide. In particular, there are multiple additional hydrogen bonds between the N-terminal end of the peptide and the IA(b) alpha chain and an extensive hydrogen bond network involving an asparagine at p7 position of the peptide and the IA(b) beta chain. By using knowledge of the shape and size of the traditional side chain binding pockets and the additional possible interactions, an IA(b) peptide-binding motif can be deduced that agrees well with the sequences of known IA(b)-binding peptides.

1. The isolated perfused rat mesenteric arterial bed was used to examine the activity of the adenine dinucleotides: beta-nicotinamide adenine dinucleotide (NAD); beta-nicotinamide adenine dinucleotide phosphate (NADP); flavin adenine dinucleotide (FAD); and of the alpha,omega-diadenosine polyphosphates: adenylyl adenosine (AP1A); P1,P2-diadenosine pyrophosphate (AP2A); P1,P3-diadenosine triphosphate (AP3A); P1,P4-diadenosine tetraphosphate (AP4A); P1,P5-diadenosine pentaphosphate (AP5A); P1,P6-diadenosine hexaphosphate (AP6A). Responses were compared with those of ADP, ATP, 2-methylthio-ATP (2-meSATP) and alpha,beta-methylene ATP (alpha,beta-meATP). 2. In basal tone preparations mono- and dinucleotides elicited vasoconstriction with the order of potency: alpha,beta-meATP>> or = AP5A>> or = AP6A>> or = AP4A>> or = 2-meSATP>>) ATP>>) ADP. The dinucleotides NAD, NADP, FAD, AP1A, AP2A and AP3A had no effect. 3. The P2X-purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (30 microM) virtually abolished vasoconstrictor responses to AP4A, AP5A and AP6A. 4. Auto- and cross-desensitization of vasoconstrictor responses to AP4A, AP5A, AP6A, ATP and alpha,beta-meATP were observed. 5. In raised tone preparations nucleotides elicited endothelium-dependent vasodilatation with the order of potency: 2-meSATP = ADP>> ATP>> AP3A>> AP2A>> AP1A = NADP = FAD>> NAD. The nucleotides AP4A, AP5A, AP6A and alpha,beta-meATP had no vasodilator effects. 6. It is concluded that the alpha,omega-adenine dinucleotides AP4A, AP5A and AP6A elicit vasoconstriction, but not vasodilatation, in the rat mesenteric arterial bed via P2x-purinoceptors. In contrast, the dinucleotides NADP, FAD, AP1A, AP2A and AP3A elicit vasodilatation, but not vasoconstriction, via endothelial P2Y-purinoceptors. 7. It is suggested that there is a crucial relationship between the structure of the alpha,omega-diadenosine polyphosphates and their activity at P2X- and P2Y-purinoceptors with a pivotal role played by the polyphosphate chain. Molecules with four or more phosphates are vasoconstrictors, while those with three or less phosphates are vasodilators.

Single crystals of the self-complementary octadeoxyribonucleotide d(GCCCGGGC) have been analysed by X-ray diffraction methods at a resolution of 1.8 A. The tetragonal unit cell of space group P4(3)2(1)2 has dimensions of a = 43.25 A and c = 24.61 A and contains eight strands of the oligonucleotide. The structure was refined by standard crystallographic techniques to an R factor of 17.1% using 1359 3 sigma structure factor observations. Two strands of the oligonucleotide are related by the crystallographic dyad axis to form a DNA helix in the A conformation. The d(GCCCGGGC) helix is characterized by a wide open major groove, a near perpendicular orientation of base pairs to the helix axis and an unusually small average helix twist angle of 31.3 degrees indicating a slightly underwound helix with 11.5 base pairs per turn. Extensive cross-strand stacking between guanine bases at the central cytosine-guanine step is made possible by a number of local conformational adjustments including a fully extended sugar-phosphate backbone of the central guanosine nucleotide.

Staphylococcal pathogenicity islands (SaPIs) are the prototypical members of a widespread family of chromosomally located mobile genetic elements that contribute substantially to intra- and interspecies gene transfer, host adaptation, and virulence. The key feature of their mobility is the induction of SaPI excision and replication by certain helper phages and their efficient encapsidation into phage-like infectious particles. Most SaPIs use the headful packaging mechanism and encode small terminase subunit (TerS) homologs that recognize the SaPI-specific pac site and determine SaPI packaging specificity. Several of the known SaPIs do not encode a recognizable TerS homolog but are nevertheless packaged efficiently by helper phages and transferred at high frequencies. In this report, we have characterized one of the non-terS-coding SaPIs, SaPIbov5, and found that it uses two different, undescribed packaging strategies. SaPIbov5 is packaged in full-sized phage-like particles either by typical pac-type helper phages, or by cos-type phages--i.e., it has both pac and cos sites--a configuration that has not hitherto been described for any mobile element, phages included--and uses the two different phage-coded TerSs. To our knowledge, this is the first example of SaPI packaging by a cos phage, and in this, it resembles the P4 plasmid of Escherichia coli. Cos-site packaging in Staphylococcus aureus is additionally unique in that it requires the HNH nuclease, carried only by cos phages, in addition to the large terminase subunit, for cos-site cleavage and melting.

The X-ray crystallographic structure of nucleoside diphosphate (NDP) kinase from Myxococcus xanthus has been determined using multiple isomorphous replacement techniques and refined at 2.0 A resolution to a crystallographic R-factor of 0.17. This is the first report of the structure of an enzymatically active NDP kinase and of the enzyme with a bound nucleotide. The structure has been determined in P4(3)2(1)2 and I222 crystal forms. The enzyme monomer consists of a four-stranded antiparallel beta-sheet. The surfaces of the sheet are partially covered with five helical segments. There are two protein molecules in the asymmetric unit of the tetragonal crystal form. They form a dimer with an extensive interface in which 1092 A2 per monomer is buried. The majority of the contact area in the dimer interface is between hydrophobic or aromatic residues. Two dimers are related by a crystallographic 2-fold axis to yield a tetramer. This tetramer is also present in the orthorhombic crystals; however, in this case, the 222 symmetry is entirely crystallographic. Upon tetramer formation, an additional 473 A2 of solvent-accessible surface area from each monomer becomes buried. The interface between dimers in the tetramer is stabilized by salt bridges. Equilibrium sedimentation studies are consistent with the enzyme being a tetramer in solution. The structure of a complex of adenosine diphosphate (ADP) with the enzyme was determined and reveals that most of the nucleotide interactions with the protein are with the pyrophosphate and ribose groups, while the base has no hydrogen bonds with the protein and interacts only by stacking with the side chain of Phe59. The Mg2+ interacts with the pyrophosphate of the ADP and via a solvent molecule with the side chain of the conserved Asp120 residue. The mode of interaction with the nucleotide is novel, with the nucleotide binding at the side of the beta-sheet. The structures of the nucleotide in crystals grown in the presence or absence of Mg2+ are essentially identical. In addition, the phosphotransfer reaction from adenosine triphosphate (ATP) to the enzyme can occur without Mg2+. This suggests that only the second step of the reaction in which the enzyme transfers the phosphate to a nucleoside diphosphate acceptor is significantly catalyzed by the metal.

Merbarone has previously been shown to have antitumor activity of unknown mechanism in P388 and L1210 tumor models (A. D. Brewer et al., Biochem. Pharmacol., 34:2047-2050, 1985) and is currently undergoing Phase I clinical trials. Here we report that merbarone is an inhibitor of topoisomerase II. Merbarone inhibited purified mammalian topoisomerase II with a 50% inhibitory concentration of 20 microM, as assessed by ATP-dependent unknotting of P4 phage DNA or relaxation of supercoiled pBR322 plasmid. In contrast to the type II enzyme, inhibition of catalytic activity of topoisomerase I required about 10-fold higher concentrations of merbarone, with a 50% inhibitory concentration of approximately 200 microM. Unlike epipodophyllotoxin analogues and certain DNA intercalative agents which stabilize the topoisomerase II-DNA "cleavable complex," merbarone did not cause detectable topoisomerase II-induced DNA cleavage. Furthermore, merbarone inhibited the production by amsacrine or teniposide of topoisomerase II-associated DNA strand breaks; under identical conditions novobiocin did not decrease these breaks, setting merbarone apart from a novobiocin-like class of topoisomerase II inhibitor. In L1210 cells, merbarone produced only small numbers of protein-associated DNA strand breaks, and only at very high concentrations. Merbarone reduced in a concentration-dependent manner the number of amsacrine- or teniposide-stimulated protein-associated DNA strand breaks in L1210 cells or their isolated nuclei. The data suggest that merbarone represents a novel type of topoisomerase II inhibitor.

A dominant epitope within the human herpesvirus 8 (HHV8) ORF 65-encoded protein was mapped to an 8-amino-acid (aa) sequence (RKPPSGKK [aa 162 to 169]) by an amino acid replacement method. Using a 14-aa peptide (P4) encompassing this epitope as the antigen, we developed an enzyme immunoassay for HHV8 antibodies. The presence of P4 antibodies in a panel of 61 human serum specimens was highly correlated with biopsy-confirmed Kaposi's sarcoma. The homologous Epstein-Barr virus peptide derived from BFBR3-encoded protein did not interfere with the assay, suggesting that P4 is specific for HHV8.

SUMMARY Taxonomic relationship: Cauliflower mosaic virus (CaMV) is the type member of the Caulimovirus genus in the Caulimoviridae family, which comprises five other genera. CaMV replicates its DNA genome by reverse transcription of a pregenomic RNA and thus belongs to the pararetrovirus supergroup, which includes the Hepadnaviridae family infecting vertebrates. Physical properties: Virions are non-enveloped isometric particles, 53 nm in diameter (Fig. 1). They are constituted by 420 capsid protein subunits organized following T= 7 icosahedral symmetry (Cheng, R.H., Olson, N.H. and Baker, T.S. (1992) Cauliflower mosaic virus: a 420 subunit (T= 7), multilayer structure. Virology, 16, 655-668). The genome consists of a double-stranded circular DNA of approximately 8000 bp that is embedded in the inner surface of the capsid. Viral proteins: The CaMV genome encodes six proteins, a cell-to-cell movement protein (P1), two aphid transmission factors (P2 and P3), the precursor of the capsid proteins (P4), a polyprotein precursor of proteinase, reverse transcriptase and ribonuclease H (P5) and an inclusion body protein/translation transactivator (P6). Hosts: The host range of CaMV is limited to plants of the Cruciferae family, i.e. Brassicae species and Arabidopsis thaliana, but some viral strains can also infect solanaceous plants. In nature, CaMV is transmitted by aphids in a non-circulative manner.

Metabolism of inositol 1,4,5-trisphosphate (I(1,4,5)P3) results in the production of diverse arrays of inositol polyphosphates (IPs), such as IP4, IP5, IP6) and PP-IP5. Insights into their synthesis in metazoans are reported here through molecular studies in the fruit fly, Drosophila melanogaster. Two I(1,4,5)P3 kinase gene products are implicated in initiating catabolism of these important IP regulators. We find dmIpk2 is a nucleocytoplasmic 6-/3-kinase that converts I(1,4,5)P3 to I(1,3,4,5,6)P5, and harbors 5-kinase activity toward I(1,3,4,6)P4, and dmIP3K is a 3-kinase that converts I(1,4,5)P3 to I(1,3,4,5)P4. To assess their relative roles in the cellular production of IPs we utilized complementation analysis, RNA interference, and overexpression studies. Heterologous expression of dmIpk2, but not dmIP3K, in ipk2 mutant yeast recapitulates phospholipase C-dependent cellular synthesis of IP6. Knockdown of dmIpk2 in Drosophila S2 cells and transgenic flies results in a significant reduction of IP6 levels; whereas depletion of dmIP3K, either alpha or beta isoforms or both, does not decrease IP6 synthesis but instead increases its production, possibly by expanding I(1,4,5)P3 pools. Similarly, knockdown of an I(1,4,5)P3 5-phosphatase results in significant increase in dmIpk2/dmIpk1-dependent IP6 synthesis. IP6 production depends on the I(1,3,4,5,6)P5 2-kinase activity of dmIpk1 and is increased in transgenic flies overexpressing dmIpk2. Our studies reveal that phosphatase and kinase regulation of I(1,4,5)P3 metabolic pools directly impinge on higher IP synthesis, and that the major route of IP6 synthesis depends on the activities of dmIpk2 and dmIpk1, but not dmIP3K, thereby challenging the role of IP3K in the genesis of higher IP messengers.

Capsids of equine infectious anemia virus have been isolated as cone-shaped particles 60 x 120 nm in size. Detergent treatment of whole virus followed by two cycles of rate-zonal centrifugation in Ficoll produces these capsids in a yield of approximately 10%. The major protein components are the gag-encoded p11 nucleocapsid protein and p26 capsid protein, which are present in equimolar amounts. Substantial cleavage of p11 to p6 and p4 can be observed under conditions where the viral protease packaged in the capsid is enzymatically active.

2,3-Dihydroxybiphenyl 1,2-dioxygenase, an enzyme of the biphenyl biodegradation pathway that cleaves the first of the aromatic rings, was purified to apparent homogeneity from Pseudomonas sp. strain LB400 that had been engineered to hyperexpress the bphC gene. The enzyme had a subunit molecular mass of 33.2 kDa as determined by SDS-polyacrylamide electrophoresis. Kinetic studies indicate a KM of 7 +/- 1 microM for 2,3-dihydroxybiphenyl. The enzyme is strongly inhibited by substrate (Kss = 300 +/- 10 microM). Catechol, 3-methylcatechol, and 4-methylcatechol were cleaved less efficiently and showed weaker substrate inhibition. 3,4-Dihydroxybiphenyl was not a substrate for the enzyme. Ammonium sulfate and polyethylene glycol 6000 were used as precipitants to obtain two different crystal forms. Crystals grown from ammonium sulfate and polyethylene glycol 6000 had space groups of P4(2)2(1)2 and I222, respectively. Electron microscopy indicates that the enzyme is an octamer (265 kDa) consisting of subunits arranged in two planar tetramers in a staggered conformation.

The calyx of Held synapse of the medial nucleus of the trapezoid body functions as a relay synapse in the auditory brainstem. In vivo recordings have shown that this synapse displays low release probability and that the average size of synaptic potentials does not depend on recent history. We used a ventral approach to make in vivo extracellular recordings from the calyx of Held synapse in rats aged postnatal day 4 (P4) to P29 to study the developmental changes that allow this synapse to function as a relay. Between P4 and P8, we observed evidence for the presence of large short-term depression, which was counteracted by short-term facilitation at short intervals. Major changes occurred in the last few days before the onset of hearing for air-borne sounds, which happened at P13. The bursting pattern changed into a primary-like pattern, the amount of depression and facilitation decreased strongly, and the decay of facilitation became much faster. Whereas short-term plasticity was the most important cause of variability in the size of the synaptic potentials in immature animals, its role became minor around hearing onset and afterward. Similar developmental changes were observed during stimulation experiments both in brain slices and in vivo following cochlear ablation. Our data suggest that the strong reduction in release probability and the speedup of the decay of synaptic facilitation that happen just before hearing onset are important events in the transformation of the calyx of Held synapse into an auditory relay synapse.

Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.

The hymenopteran wasp Leptopilina boulardi (Figitidae) is a larval solitary parasitoid of Drosophila larvae of the melanogaster sub-group. The factors used by parasitoid females to prevent encapsulation of their eggs by the host are localized in the female long gland and reservoir. We report here the physiological effects of these factors on host haemocytes using in vivo injection experiments. The total number of haemocytes, the number of plasmatocytes and the number of crystal cells were not modified by injection of long gland extracts. In contrast, long gland extracts either from virulent or avirulent strains had a significant effect on the lamellocyte number. Compared to the Ringer control, the avirulent long gland products induced an increase of the lamellocyte number while virulent extracts induced a drastic decrease together with an alteration of the morphology of these cells. Interestingly, changes in the lamellocyte morphology were also observed following injection of the P4 protein, a major component of L. boulardi female long glands that displays a strong immune suppressive effect on Drosophila larvae. The implication of the P4 protein in suppressing the host cellular immunity is discussed in correlation with its predicted molecular function as a Rho-GAP protein.

Transcription of the genome of Bacillus subtilis phage phi 29 is tightly controlled, taking place in two stages, early and late. We have analyzed the abundance of the transcripts produced from each viral promoter throughout the infection cycle. We compare the relative strength of each promoter, as well as get a better understanding of the regulatory events, finding a new promoter regulated by the viral protein p4. The two strong early promoters, A2b and A2c, responsible for the expression of genes 6 to 1, are coordinately repressed by the viral protein p4, although repression is not complete: both promoters are still active at late times of infection. Since repression by protein p4 was very efficient in vitro, and affects its own synthesis, it is likely that this protein is produced in limiting amounts, not being bound to all viral DNA molecules present in the cell at a given time. Protein p4, also known to activate the late promoter responsible for the expression of all the structural and morphogenetic genes, is the key regulator of phage phi 29 development.

We report here that disruption of function of the ω-3 FATTY ACID DESATURASE7 (FAD7) enhances plant defenses against aphids. The suppressor of prosystemin-mediated responses2 (spr2) mutation in tomato (Solanum lycopersicum), which eliminates the function of FAD7, reduces the settling behavior, survival, and fecundity of the potato aphid (Macrosiphum euphorbiae). Likewise, the antisense suppression of LeFAD7 expression in wild-type tomato plants reduces aphid infestations. Aphid resistance in the spr2 mutant is associated with enhanced levels of salicylic acid (SA) and mRNA encoding the pathogenesis-related protein P4. Introduction of the Naphthalene/salicylate hydroxylase transgene, which suppresses SA accumulation, restores wild-type levels of aphid susceptibility to spr2. Resistance in spr2 is also lost when we utilize virus-induced gene silencing to suppress the expression of NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1), a positive regulator of many SA-dependent defenses. These results indicate that FAD7 suppresses defenses against aphids that are mediated through SA and NPR1. Although loss of function of FAD7 also inhibits the synthesis of jasmonate (JA), the effects of this desaturase on aphid resistance are not dependent on JA; other mutants impaired in JA synthesis (acx1) or perception (jai1-1) show wild-type levels of aphid susceptibility, and spr2 retains aphid resistance when treated with methyl jasmonate. Thus, FAD7 may influence JA-dependent defenses against chewing insects and SA-dependent defenses against aphids through independent effects on JA synthesis and SA signaling. The Arabidopsis (Arabidopsis thaliana) mutants Atfad7-2 and Atfad7-1fad8 also show enhanced resistance to the green peach aphid (Myzus persicae) compared with wild-type controls, indicating that FAD7 influences plant-aphid interactions in at least two plant families.