A set of plasmids that contain fragments of the bacteriophage P4 genome has been constructed by deleting portions of a P4-ColE1 hybrid. A P4 genetic map has been established and related to the physical map by examining the ability of these plasmids to rescue various P4 mutations. The P4 virl mutation and P4 genes involved in DNA replication (alpha), activation of P2 helper genes (delta and epsilon), polarity suppression (psu) and head size determination (sid) have been mapped, as has the region responsible for synthesis of a nonessential P4 protein. One of the deleted plasmids contains only 5900 base pairs (52%) of P4 but will form plaques if additional DNA is added to increase its total size to near that of P4. This plasmid is also unique in that it will not form stable associations with P2 lysogens of E. coli which are recA+. P4 alpha mutants can be suppressed as a result of replication under control of the ColE1 part of the hybrid.

Epidermal growth factor (EGF) treatment of A-431 cells induces a biphasic increase in the levels of inositol phosphates. The growth factor produces an initial, rapid increase in the level of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) due to hydrolysis of phosphatidyl-inositol-4,5-bisphosphate (Wahl, M., Sweatt, J. D., and Carpenter, G. (1987) Biochem. Biophys. Res. Commun. 142, 688-695). The level of inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) also rises rapidly in response to treatment with EGF. The initial formation (less than 1 min) of Ins-1,4,5-P3 and Ins-1,3,4,5-P4 does not require Ca2+ present in the culture medium. However, the addition of Ca2+ to the medium at levels of 100 microM or greater potentiates the growth factor-stimulated increases in the levels of all inositol phosphates at later times after EGF addition (1-60 min). The data suggest that EGF-receptor complexes initially stimulate the enzyme phospholipase C in a manner that is independent of an influx of extracellular Ca2+. The presence of Ca2+ in the medium allows prolonged growth factor activation of phospholipase C. Treatment of A-431 cells with Ca2+ ionophores (A23187 and ionomycin) did not mimic the activity of EGF in producing a rapid increase in the formation of the Dowex column fraction containing Ins-1,4,5-P3, Ins-1,3,4,5-P4, and inositol 1,3,4-trisphosphate (InsP3). However, the initial EGF-stimulated formation of inositol phosphates was substantially diminished in cells loaded with the Ca2+ chelator Quin 2/AM. EGF receptor occupancy studies indicated that maximal stimulation of InsP3 accumulation by EGF requires nearly full (75%) occupancy of available EGF binding sites, while half-maximal stimulation requires 25% occupancy. 12-O-Tetradecanoylphorbol-13-acetate (TPA), an exogenous activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), causes a dramatic, but transient, inhibition of the EGF-stimulated formation of inositol phosphates. Tamoxifen and sphingosine, reported pharmacologic inhibitors of protein kinase C activity, potentiate the capacity of EGF to induce formation of inositol phosphates. Neither TPA nor tamoxifen significantly affects the 125I-EGF binding capacity of A-431 cells; however, TPA appeared to enhance internalization of the ligand. Ligand occupation of the EGF receptor on the A-431 cell appears to initiate a complex signaling mechanism involving production of intracellular messengers for Ca2+ mobilization and activation of protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)

Lysogens of Shigella flexneri harbouring the temperate bacteriophage, Sf6, have been previously shown to undergo a serotype conversion due to O-acetylation of the O-antigen of the lipopolysaccharide. A partial physical map of the phage genome has been constructed. Analysis of the phage DNA suggests that the phage packages by a headful mechanism and that the mature DNA molecules are terminally redundant. Cloning of the PstI fragments of Sf6 enabled the region encoding the serotype conversion to be localized, showing that this was clearly phage-encoded. The gene was further localized by mutagenesis with Tn5 and the nucleotide sequence of the entire 2693-bp PstI fragment was determined. Two major open reading frames (ORFs) were found capable of encoding proteins of 44.1 and 37.2 kDa. The latter corresponds to the O-antigen acetylase and its gene has been designated oac. The oac gene is capable of converting Sh. flexneri serotypes X, Y, 1a and 4a to 3a, 3b, 1b and 4b, respectively. The Oac protein bears a high degree of homology to the NodX protein of Rhizobium leguminosarum suggesting that it, too, may be a sugar acetylase. The second ORF immediately upstream from oac corresponds to the bacteriophage Sf6 integrase responsible for chromosomal integration and is highly homologous to the integrases of Escherichia coli bacteriophages P4 and phi 80, but less closely related to those of P1, P2, P22, 186 and lambda.

In this study we report the complete nucleotide sequence and genetic organization of the she pathogenicity island (PAI) of Shigella flexneri 2a strain YSH6000T. The 46 603 bp she PAI is situated adjacent to the 3' terminus of the pheV tRNA gene and includes an imperfect direct repeat of the 3'-terminal 22 bp of the pheV gene at the right boundary of the PAI. The she PAI carries a bacteriophage P4-like integrase gene within the pheV -proximal boundary of the PAI, intact and truncated mobile genetic elements, plasmid-related sequences, open reading frames exhibiting high sequence similarity to those found on the locus of enterocyte effacement (LEE) PAI of enterohemorrhagic Escherichia coli (EHEC), and the SHI-2 PAI of S. flexneri and several other open reading frames of unknown function. The she PAI also encodes two autotransporter proteins, including SigA, a cytopathic protease that contributes to intestinal fluid accumulation and Pic, a protease with mucinase, and hemagglutinin activities. In addition, an open reading frame (orf) termed sap, has high sequence similarity to the gene encoding Antigen 43, a surface-located autotransporter protein of E. coli. The ShET1 enterotoxin genes, associated predominantly with S. flexneri 2a strains, are also located on the she PAI.

The Escherichia coli cydAB operon, encoding the subunits of the high-affinity cytochrome d oxidase, is maximally transcribed in microaerobiosis as a result of the combined action of the oxygen-responsive regulators Fnr and ArcA. Here, we report that the histone-like protein H-NS is an aerobic repressor of cydAB expression. ArcA is shown to antagonize H-NS action to render cydAB expression insensitive to H-NS repression in anaerobiosis. The targets for H-NS-mediated aerobic repression are the four oxygen-regulated promoters, designated P1, P2, P3 and P4. H-NS control is the result of H-NS binding to an extended region within the cydAB promoter element, including sequences upstream from and overlapping the four regulated promoters. We propose a regulatory model in which oxygen control of cydAB transcription is mediated by three alternative protein-DNA complexes that are assembled sequentially on the promoter region as the cells are shifted from aerobic to microaerobic and to anaerobic conditions. According to this model, ArcA-P plays a central role in cydAB regulation by antagonizing H-NS repression of cydAB transcription when oxygen becomes limiting. This allows peak gene expression and subsequent repression by Fnr under fully anaerobic conditions.

Five dengue (DEN) virus-specific R5F2R4 peptide-conjugated phosphorodiamidate morpholino oligomers (P4-PMOs) were evaluated for their ability to inhibit replication of DEN virus serotype 2 (DEN-2 virus) in mammalian cell culture. Initial growth curves of DEN-2 virus 16681 were obtained in Vero cells incubated with 20 microM P4-PMO compounds. At 6 days after infection, a P4-PMO targeting the 3'-terminal nucleotides of the DEN-2 virus genome and a random-sequence P4-PMO showed relatively little suppression of DEN-2 virus titer (0.1 and 0.9 log10, respectively). P4-PMOs targeting the AUG translation start site region of the single open reading frame and the 5' cyclization sequence region had moderate activity, generating 1.6- and 1.8-log10 reductions. Two P4-PMO compounds, 5'SL and 3'CS (targeting the 5'-terminal nucleotides and the 3' cyclization sequence region, respectively), were highly efficacious, each reducing the viral titer by greater than 5.7 log10 compared to controls at 6 days after infection with DEN-2 virus. Further experiments showed that 5'SL and 3'CS inhibited DEN-2 virus replication in a dose-dependent and sequence-specific manner. Treatment with 10 microM 3'CS reduced the titers of all four DEN virus serotypes, i.e., DEN-1 (strain 16007), DEN-2 (16681), DEN-3 (16562), and DEN-4 (1036) viruses by over 4 log10, in most cases to below detectable limits. The extent of 3'CS efficacy was affected by the timing of compound application in relation to viral infection of the cells. The 5'SL and 3'CS P4-PMOs did not suppress the replication of West Nile virus NY99 in Vero cells. These data indicate that further evaluation of the 5'SL and 3'CS compounds as potential DEN virus therapeutics is warranted.

To identify signals that convey connexin oligomerization compatibility, we have aligned amino-acid sequences of alpha and beta group connexins (Cx) and compared the physico-chemical properties of each homologous amino-acid residue. Four positions were identified that consistently differed between alpha and beta-type connexins; two are located in the N-terminal domain (P1 and P2, corresponding to residues 12 and 13 of the Cx43 sequence), and two in the third trans-membrane-spanning domain TM3 (P3 and P4, corresponding to residues 152 and 153 of the Cx43 sequence). Replacement of each of these residues in Cx43 (an alpha-type connexin) with the corresponding residues of Cx32 (a beta-type connexin) resulted in the assembly of all variants into gap junctions; however, only the P4 variant was functional, as indicated by lucifer yellow dye transfer assays. The other three variants exerted a moderate to severe dose-dependent, dominant-negative effect on co-expressed wild-type (wt) Cx43 channel activity. Moreover, a significant dose-dependent, trans-dominant inhibition of channel activity was observed when either one of the N-terminal variants was co-expressed with wt Cx32. Assembly analyses indicated that dominant and trans-dominant inhibitory effects appeared to be based on the oligomerization of wt and variant connexins into mixed connexons. Interestingly, the identified N-terminal amino acids coincide with the position of naturally occurring, disease-causing missense mutations of several beta-connexin genes (Cx26, Cx30, Cx31, Cx32). Our results demonstrate that three of the identified discriminative amino-acid residues (positions 12, 13 and 152) are crucial for Cx43 channel function and suggest that the N-terminal amino-acid residues at position 12/13 are involved in the oligomerization compatibility of alpha and beta connexins.

Antigen receptor-mediated production of inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) in lymphocytes triggers the release of Ca2+ from intracellular stores; this release of Ca2+ results in the opening of store-operated Ca2+ channels in the plasma membrane. Here we report that mice lacking Ins(1,4,5)P3 3-kinase B (Itpkb), which converts Ins(1,4,5)P3 to inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), had impaired B lymphocyte development and defective immunoglobulin G3 antibody responses to a T lymphocyte-independent antigen. Itpkb-deficient B lymphocytes had the phenotypic and functional features of tolerant B lymphocytes and showed enhanced activity of store-operated Ca2+ channels after B lymphocyte receptor stimulation, which was reversed by the provision of exogenous Ins(1,3,4,5)P4. Our data identify Itpkb and its product Ins(1,3,4,5)P4 as inhibitors of store-operated Ca2+ channels and crucial regulators of B cell selection and activation.

Members of the P(4) subfamily of P-type ATPases are believed to catalyze flipping of phospholipids across cellular membranes, in this way contributing to vesicle biogenesis in the secretory and endocytic pathways. P(4)-ATPases form heteromeric complexes with Cdc50-like proteins, and it has been suggested that these act as beta-subunits in the P(4)-ATPase transport machinery. In this work, we investigated the role of Cdc50-like beta-subunits of P(4)-ATPases for targeting and function of P(4)-ATPase catalytic alpha-subunits. We show that the Arabidopsis P(4)-ATPases ALA2 and ALA3 gain functionality when coexpressed with any of three different ALIS Cdc50-like beta-subunits. However, the final cellular destination of P(4)-ATPases as well as their lipid substrate specificity are independent of the nature of the ALIS beta-subunit they were allowed to interact with.

The spatial and temporal expressions of Muc-1, selected integrin subunits, and extracellular matrix components in porcine uterine epithelium from estrous (Days 0, 4, 8, 10-15) and early pregnant (Days 10-15 of pregnancy) gilts and from steroid-treated ovariectomized gilts were analyzed using indirect immunofluorescence analyses on cryosectioned tissues to identify potential components of uterine receptivity. Integrin subunit and extracellular matrix expressions were also examined in Day 11-15 conceptuses. Intense Muc-1 staining was detected on apical uterine epithelium on Day 0 but was absent by Day 10 in both cyclic and pregnant gilts. The result of estrogen treatment (E2; 100 micrograms/day for 10 days) was similar to that of the corn oil vehicle control, while treatment with progesterone (P4; 200 mg/day for 10 days) or E2 + P4 decreased Muc-1 staining in ovariectomized gilts. Immunostaining performed with antibodies directed against integrin subunits (alpha 1, alpha 3, alpha 4, alpha 5, alpha v, beta 1, and beta 3) in uterine epithelium revealed low (integrin subunits alpha 1 alpha 3), high (integrin subunits alpha v and beta 3), or modulated (integrin subunits alpha 4, alpha 5, and beta 1) expression, with the lowest expression on Day 0 and maximum expression by Days 10-15. Additionally, no differences due to pregnancy status were detected in staining of uterine epithelium on Days 10-15. Uterine epithelium from steroid-treated ovariectomized gilts had low expression of alpha 4, alpha 5, and beta 1 subunits in the presence of E2 that increased in response to P4 and E2 + P4 treatments. The expression of integrin subunits alpha 3, alpha v and beta 3 was not affected by sex steroids. Trophectoderm also expressed alpha 1, alpha 4, alpha 5, alpha v, beta 1, and beta 3, integrin subunits. Extracellular matrix constituents (fibronectin, vitronectin, laminin, and collagen type IV) were also examined. Fibronectin and vitronectin were present on trophectoderm, but only vitronectin was detected on uterine epithelium. The alpha 4, alpha 5, alpha v, beta 1, and beta 3 integrin subunits, vitronectin, and fibronectin were detected at sites of attachment between uterine epithelial cells and trophectoderm on Days 12-15 of pregnancy. These studies indicate that down-regulation of Muc-1 coincides with the transition of the prereceptive uterus to the receptive uterus. Additionally, the expression of alpha 4, alpha 5, alpha v, beta 1, and beta 3 integrin subunits along with the extracellular matrix components of fibronectin and vitronectin correlates with the time of implantation in swine.

RNA molecules commonly consist of helical regions separated by internal loops, and in many cases these internal loops have been found to assume stable structures. We have examined the function and dynamics of an internal loop, J5/5a, that joins the two halves of the P4-P6 domain of the Tetrahymena self-splicing group I intron. P4-P6 RNAs with mutations in the J5/5a region showed nondenaturing gel electrophoretic mobilities and levels of Fe(II)-EDTA cleavage protection intermediate between those of wild-type RNA and a mutant incapable of folding into the native P4-P6 tertiary structure. Mutants with the least structured J5/5a loops behaved the most like wild-type P4-P6, and required smaller amounts of Mg2+ to rescue folding. The activity of reconstituted introns containing mutant P4-P6 RNAs correlated similarly with the nature of the J5/5a mutation. Our results suggest that, in solution, the P4-P6 RNA is in a two-state equilibrium between folded and unfolded states. We conclude that this internal loop mainly acts as a flexible hinge, allowing the coaxially stacked helical regions on either side of it to interact via specific tertiary contacts. To a lesser extent, the specific bases within the loop contribute to folding. Furthermore, it is crucial that the junction remain unstructured in the unfolded state. These conclusions cannot be derived from a simple examination of the P4-P6 crystal structure (Cate JH et al., 1996, Science 273:1678-1685), showing once again that structure determination must be supplemented with mutational and thermodynamic analysis to provide a complete picture of a folded macromolecule.

Low bone mass is known to be associated with an increased risk of fractures. Osteoporosis prevention by maximizing bone mass will be crucial and requires a better knowledge of bone mass acquisition during adolescence. Bone mass was assessed in 574 healthy volunteer females aged 10-24 years. Spine bone mineral density (BMD) in anteroposterior (AP L2-4) and lateral (LAT L3) views was measured using dual-energy X-ray absorptiometry (DXA) and AP bone mineral content (BMC) was calculated. At the same time, spine AP-BMD (L2-4) was evaluated in 333 normal menstruating women, aged 27-47 years. Bone values, osteocalcin and IGF-1 serum concentrations were correlated with chronological age, skeletal age, pubertal stages and time after menarche. In this cross-sectional study, AP- and LAT-BMD and BMC increased dramatically between skeletal ages 10 and 14 or until the first year after menarche. Between 14 and 17 skeletal years of age, AP-BMD and BMC increased moderately, whereas LAT-BMD remained unchanged. After skeletal age 17, or the fourth year after menarche, there was no significant increase in BMD or BMC, and their values did not differ from those of menstruating women. A serum osteocalcin peak was observed at skeletal ages 11-12 or at stage P3, whereas IGF-1 peaked at 13-14 skeletal years of age or at P4 and the first year after menarche. Eighty-six per cent of the adult bone mass of the spine is acquired before skeletal age 14 or the second year after menarche; therefore osteoporosis prevention programs will be particularly effective before that age.

We examined the promoter selectivity of RNA polymerase (RNAP) from Streptomyces coelicolor at two growth phases by in vitro transcription. Distinct sets of promoters were preferentially recognized by either exponential or stationary phase RNAP. No change in molecular weight or net charge of the core subunits was observed, suggesting that the associated specificity factors determined phase-specific promoter selectivity of the holoenzyme. Five different specificity factors and their cognate promoters were identified by in vitro holoenzyme reconstitution and transcription assays. sigma66 (sigma hrdB) and sigma46 (sigma hrdD) recognized promoters (rrnD p2 and dagA p4 for sigma66, actII-orf4 p and whiB p2 for sigma46) preferentially transcribed by the exponential phase RNAP. sigma52 recognized promoters (dagA p3 and actIII px1) preferentially transcribed by the stationary phase RNAP. Sigma28 (sigma sigE) recognized promoters (hrdD p1, whiB p1 and dagA p2) transcribed equally by both RNAPs. A novel 31 kDa specificity factor recognized actIII px2, glnR p2 and hrdD p2 promoters preferentially transcribed by the stationary phase RNAP. This factor was isolated from the stationary phase RNAP and reconstituted holoenzyme in vitro as a sigma factor. The N-terminal sequence suggests that it is a novel factor. By examining phase-specific promoter recognition pattern we can predict that holoenzyme Esigma52 and Esigma31 activities are higher in the stationary phase, whereas Esigma66 and Esigma46activities are higher in the exponential phase. Possible promoter sequences recognized by some of these sigma factors were suggested.

The genetic heterogeneity and antifungal susceptibility patterns of Candida parapsilosis isolated from blood cultures of patients were investigated in this study. Randomly amplified polymorphic DNA (RAPD) analysis generated 5 unique profiles from 42 isolates. Based on the major DNA fragments of the RAPD profiles, the isolates were identified as RAPD type P1 (29 isolates), P2 (6 isolates), P3 (4 isolates), P4 (2 isolates) and P5 (1 isolate). Sequence analysis of the internal transcribed spacer (ITS) gene of the isolates identified RAPD type P1 as C. parapsilosis, P2 and P3 as Candida orthopsilosis, P4 as Candida metapsilosis, and P5 as Lodderomyces elongisporus. Nucleotide variations in ITS gene sequences of C. orthopsilosis and C. metapsilosis were detected. Antifungal susceptibility testing using Etests showed that all isolates tested in this study were susceptible to amphotericin B, fluconazole, ketoconazole, itraconazole and voriconazole. C. parapsilosis isolates exhibited higher MIC(50) values than those of C. orthopsilosis for all of the drugs tested in this study; however, no significant difference in the MICs for these two Candida species was observed. The fact that C. orthopsilosis and C. metapsilosis were responsible for 23.8 and 4.8 % of the cases attributed to C. parapsilosis bloodstream infections, respectively, indicates the clinical relevance of these newly described yeasts. Further investigations of the ecological niche, mode of transmission and virulence of these species are thus essential.

The mechanism by which inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5, 6)P4) regulates chloride (Cl-) secretion was evaluated in the colonic epithelial cell line T84 using whole cell voltage clamp techniques. Our studies focused on the calcium-dependent chloride conductance (gClCa) that was activated either by mobilizing intracellular calcium (Cai) stores with thapsigargin or by introduction of the autonomous, autophosphorylated calmodulin-dependent protein kinase II (CaMKII) into the cell via the patch pipette. Basal concentrations of Ins(3,4,5,6)P4 (1 microM) present in the pipette solution had no significant effect on Cl- current; however, as the concentration of the polyphosphate was increased there was a corresponding reduction in anion current, with near complete inhibition at 8-10 microM Ins(3,4,5,6)P4. Corresponding levels are found in cells after sustained receptor-dependent activation of phospholipase C. The Ins(3,4,5, 6)P4-induced inhibition of gClCa was isomer specific; neither Ins(1, 3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, nor Ins(1,3,4,5,6)P5 induced current inhibition at concentrations of up to 100 microM. Annexin IV also plays an inhibitory role in modulating gClCa in T84 cells. When 2 microM annexin IV was present in the pipette solution, a concentration that by itself has no effect on gClCa, the potency of Ins(3,4,5,6)P4 was approximately doubled. The combination of Ins(3,4,5,6)P4 and annexin IV did not alter the in vitro activity of CaMKII. These data demonstrate that Ins(3,4,5,6)P4 is an additional cellular signal that participates in the control of salt and fluid secretion, pH balance, osmoregulation, and other physiological activities that depend upon gClCa activation. Ins(3,4,5,6)P4 metabolism and action should also be taken into account when designing treatment strategies for cystic fibrosis.

The satellite bacteriophage P4 does not have genes coding for any major structural proteins, but assembles a capsid from the gene products of bacteriophage P2. The capsid assembled under control of P4 is smaller (45 nm) than the normal P2 capsid (60 nm). The low resolution (4.5 nm) structures of P2 and P4 capsids were determined by cryo-electron microscopy and image processing. The capsid of P2 shows T = 7 symmetry with most of the mass clustered as 12 pentamers and 60 hexamers. The P4 capsid has T = 4 symmetry with a similar distribution of mass to P2, but the hexamer geometry has changed. The major capsid protein has a two-domain structure. The major domains form the capsomers proper, while connecting domains form trivalent contacts between the capsomers. The size determination by P4 appears to function by altering hexamer geometry rather than by affecting the interdomain angle alone.

There are four transcriptional promoters present in the 5' control region of the Escherichia coli DNA topoisomerase I (topA) gene. These were identified with Bal31 nuclease-generated deletions and mapping of the 5' ends of the mRNAs with avian reverse transcriptase. Recombinant plasmids with all or some of these promoters fused to the galactokinase (galK) gene-coding region have been constructed and used to study transcription from the promoters both in vitro and in vivo. The promoter (P1) closest to the starting ATG codon has a near consensus -35 sequence (GTTGATA) but unusual -10 (CATATCG) sequence. The other three promoters (P2, P3 and P4) are clustered together 60 base-pairs further upstream. Negative DNA supercoiling is required for efficient transcription from P1, P1 + P2 + P3 + P4, P2 + P3 + P4, P3 + P4 and P4 alone. The combination of all four promoters demonstrates greater supercoiling dependence than does any of the other subsets tested.

A study was made of the fate of parental bluetongue virus (BTV) in infected cells. Within the first hour after infection, the BTV particles are converted to core particles with the loss of major capsid polypeptides P2 and P5. The particles are able to synthesize mRNA in vitro in a transcription reaction characterized by a temperature-dependent inhibition at high core concentrations. From about 6 hr after infection a second uncoating event is observed in which the 470 S core particles are converted to 390 S subcore particles. These particles are morphologically strikingly different from core particles and have a skeletonlike structure with a hexagonal profile and a side to side diameter of 40 nm. These subcore particles contain only one major structural protein, P3, and three minor proteins, P1, P4, and P6. They do, however, contain all 10 double-stranded RNA segments. The results suggest that the characteristic capsomeres on the surface of core particles are composed mainly of P7, the soluble group-specific antigen of BTV. The subcore particles are stable only at very low salt concentrations. Under these conditions no transcriptase activity can be demonstrated.

The analysis of the inositol cycle in Dictyostelium discoideum cells is complicated by the limited uptake of [3H]inositol (0.2% of the applied radioactivity in 6 h), and by the conversion of [3H]inositol into water-soluble inositol metabolites that are eluted near the position of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] on anion-exchange h.p.l.c. columns. The uptake was improved to 2.5% by electroporation of cells in the presence of [3H]inositol; electroporation was optimal at two 210 microseconds pulses of 7 kV. Cells remained viable and responsive to chemotactic signals after electroporation. The intracellular [3H]inositol was rapidly metabolized to phosphatidylinositol and more slowly to phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. More than 85% of the radioactivity in the water-soluble extract that was eluted on Dowex columns as Ins(1,4,5)P3 did not co-elute with authentic [32P]Ins(1,4,5)P3 on h.p.l.c. columns. Chromatography of the extract by ion-pair reversed-phase h.p.l.c. provided a good separation of the polar inositol polyphosphates. Cellular [3H]Ins(1,4,5)P3 was identified by (a) co-elution with authentic [32P]Ins(1,4,5)P3 and (b) degradation by a partially purified Ins(1,4,5)P3 5-phosphatase from rat brain. The chemoattractant cyclic AMP and the non-hydrolysable analogue guanosine 5'-[gamma-thio]triphosphate induced a transient accumulation of radioactivity in Ins(1,4,5)P3; we did not detect radioactivity in inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In vitro, Ins(1,4,5)P3 was metabolized to inositol 1,4- and 4,5-bisphosphate, but not to Ins(1,3,4,5)P4 or another tetrakisphosphate isomer. We conclude that Dictyostelium has a receptor- and G-protein-stimulated inositol cycle which is basically identical with that in mammalian cells, but the metabolism of Ins(1,4,5)P3 is probably different.

Type A botulinum neurotoxin (botox A) is a zinc metalloprotease that cleaves only one peptide bond in the synaptosomal protein, SNAP-25. Single-residue changes in a 17-residue substrate peptide were used to develop the first specific, competitive inhibitors of its proteolytic activity. Substrate analog peptides with P4, P3, P2' or P3' cysteine were readily hydrolyzed by the toxin, but those with P1 or P2 cysteine were not cleaved and were inhibitors. Peptides with either D- or L-cysteine as the N-terminus, followed by the last six residues of the substrate, were the most effective inhibitors, each with a Ki value of 2 microM. Elimination of the cysteine sulfhydryl group yielded much less effective inhibitors, suggesting that inhibition was primarily due to binding of the active-site zinc by the sulfhydryl group. Botox A displayed an unusual requirement for arginine as the P1' inhibitor residue, demonstrating that the S1' binding subsite of botox A is dissimilar to those of most other zinc metalloproteases. This characteristic is an important element in shaping the substrate specificity of botox A.

The periplasmic dimethyl sulfoxide reductase (DMSOR) from the photosynthetic purple bacterium Rhodobacter capsulatus functions as the terminal electron acceptor in its respiratory chain. The enzyme catalyzes the reduction of highly oxidized substrates like dimethyl sulfoxide to dimethyl sulfide. At a molybdenum redox center, two single electrons are transferred from cytochrome C556 to the substrate dimethyl sulfoxide, generating dimethyl sulfide and (with two protons) water. The enzyme was purified and crystallized in space group P4(1)2(1)2 with unit cell dimensions of a = b = 80.7 A and c = 229.2 A. The crystals diffract beyond 1.8 A with synchrotron radiation. The three-dimensional structure was solved by a combination of multiple isomorphous replacement and molecular replacement techniques. The atomic model was refined to an R-factor of 0.169 for 57,394 independent reflections. The spherical protein consists of four domains with a funnel-like cavity that leads to the freely accessible metal-ion redox center. The bis(molybdopterin guanine dinucleotide) molybdenum cofactor (1541 Da) of the single chain protein (85,033 Da) has the molybdenum ion bound to the cis-dithiolene group of only one molybdopterin guanine dinucleotide molecule. Three additional ligands, two oxo groups and the oxygen of a serine side-chain, are bound to the molybdenum ion. The second molybdopterin system is not part of the ligand sphere of the metal center with its sulfur atoms at distances of 3.5 A and 3.8 A away. It might be involved in electron shuttling from the protein surface to the molybdenum center.

Noninvasive fecal assays were used to study steroid metabolism and ovarian activity in several felid species. Using the domestic cat (Felis catus) as model, the excretory products of injected [14C]estradiol (E2) and [14C]progesterone (P4) were determined. Within 2 days, 97.0 +/- 0.6% and 96.7 +/- 0.5% of recovered E2 and P4 radioactivity, respectively, was found in feces. E2 was excreted as unconjugated estradiol and estrone (40%) and as a non-enzyme-hydrolyzable conjugate (60%). P4 was excreted primarily as non-enzyme-hydrolyzable, conjugated metabolites (78%) and as unconjugated pregnenolone epimers. A simple method for extracting fecal steroid metabolites optimized extraction efficiencies of the E2 and P4 excretion products (90.1 +/- 0.8% and 87.2 +/- 1.4%, respectively). Analysis of HPLC fractions of extracted fecal samples from the radiolabel-injected domestic cats revealed that E2 immunoreactivity coincided primarily with the unconjugated metabolized [14C]E2 peak, whereas progestogen immunoreactivity coincided with a single conjugated epimer and multiple unconjugated pregnenolone epimers. After HPLC separation, similar immunoreactive E2 and P4 metabolite profiles were observed in the leopard cat (F. bengalensis), cheetah (Acinonyx jubatus), clouded leopard (Neofelis nebulosa), and snow leopard (Panthera uncia). Longitudinal analyses demonstrated that changes in fecal E2 and P4 metabolite concentrations reflected natural or artificially induced ovarian activity. For example, severalfold increases in E2 excretion were associated with overt estrus or exogenous gonadotropin treatment, and elevated fecal P4 metabolite concentrations occurred during pregnant and nonpregnant (pseudopregnant) luteal phases. Although overall concentrations were similar, the duration of elevated fecal P4 metabolites during pseudopregnancy was approximately half that observed during pregnancy. In summary, steroid metabolism mechanisms appear to be conserved among these physically diverse, taxonomically related species. Results indicate that this hormone-monitoring approach will be extremely useful for elucidating the hormonal regulatory mechanism associated with the reproductive cycle, pregnancy, and parturition of intractable and endangered felid species.

Genetic inactivation of monoamine oxidase A (MAOA) in C3H/HeJ mice causes a complete absence of barrels in the somatosensory cortex, and similar alterations are caused by pharmacological inhibition of MAOA in wild type mice. To determine when and how MAOA inhibition affects the development of the barrel field, the MAOA inhibitor clorgyline was administered to mice of the outbred strain OF1 for various time periods between embryonic day 15 (E15) and postnatal day 7 (P7), and the barrel fields were analyzed with cytochrome oxidase and Nissl stains in P10 and adult mice. High-pressure liquid chromatography measures of brain serotonin (5-HT) showed three- to eightfold increases during the periods of clorgyline administration. Perinatal mortality was increased and weight gain was slowed between P3 and P6. Clorgyline treatments from E15 to P7 or from P0 to P7 disrupted the formation of barrels in the anterior snout representation and in parts of the posteromedial barrel subfield (PMBSF). Treatments from P0 to P4 caused similar although less severe barrel field alterations. Clorgyline treatments only during embryonic life or starting on P4 caused no detectable abnormalities. In cases with barrel field alterations, a rostral-to-caudal gradient of changes was noted: Rostral barrels of the PMBSF were most frequently fused and displayed an increased size tangentially. Thus, MAOA inhibition resulting in increased brain levels of 5-HT affects barrel development during the entire first postnatal week, with a sensitive period between P0 and P4. The rostral-to-caudal gradient of changes in the barrel field parallels known developmental gradients in the sensory periphery and in the maturation thalamocortical afferents. The observed barrel fusions could correspond to a default in the initial segregation of thalamic fibers or to a continued, exuberant growth of these fibers that overrides the tangential domain that is normally devoted to individual whiskers.

Because of the probable causal relationship between constitutive p210(bcr/abl) protein tyrosine kinase activity and manifestations of chronic-phase chronic myelogenous leukemia (CML; myeloid expansion), a key goal is to identify relevant p210 substrates in primary chronic-phase CML hematopoietic progenitor cells. We describe here the purification and mass spectrometric identification of a 155-kD tyrosine phosphorylated protein associated with src homologous and collagen gene (SHC) from p210(bcr/abl)-expressing hematopoietic cells as SHIP2, a recently reported, unique SH2-domain-containing protein closely related to phosphatidylinositol polyphosphate 5-phosphatase SHIP. In addition to an N-terminal SH2 domain and a central catalytic region, SHIP2 (like SHIP1) possesses both potential PTB(NPXY) and SH3 domain (PXXP) binding motifs. Thus, two unique 5-ptases with striking structural homology are coexpressed in hematopoietic progenitor cells. Stimulation of human hematopoietic growth factor responsive cell lines with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrate the rapid tyrosine phosphorylation of SHIP2 and its resulting association with SHC. This finding suggests that SHIP2, like that reported for SHIP1 previously, is linked to downstream signaling events after activation of hematopoietic growth factor receptors. However, using antibodies specific to these two proteins, we demonstrate that, whereas SHIP1 and SHIP2 selectively hydrolyze PtdIns(3,4,5)P3 in vitro, only SHIP1 hydrolyzes soluble Ins(1,3,4,5)P4. Such an enzymatic difference raises the possibility that SHIP1 and SHIP2 may serve different functions. Preliminary binding studies using lysates from p210(bcr/abl)-expressing cells indicate that both Ptyr SHIP2 and Ptyr SHIP1 bind to the PTB domain of SHC but not to its SH2 domain. Interestingly, SHIP2 was found to selectively bind to the SH3 domain of ABL, whereas SHIP1 selectively binds to the SH3 domain of Src. Furthermore, in contrast to SHIP1, SHIP2 did not bind to either the N-terminal or C-terminal SH3 domains of GRB2. These observations suggest (1) that SHIP1 and SHIP2 may have a different hierarchy of binding SH3 containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and (2) that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. Because recent evidence has clearly implicated both PI(3,4, 5)P3 and PI(3,4)P2 in growth factor-mediated signaling, our finding that both SHIP1 and SHIP2 are constitutively tyrosine phosphorylated in CML primary hematopoietic progenitor cells may thus have important implications in p210(bcr/abl)-mediated myeloid expansion.