Angiotensin stimulates rapid and prominent increases in inositol polyphosphates and their metabolites in bovine glomerulosa cells labeled with [3H]inositol. In addition to the early formation of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4-trisphosphate (Ins-1,3,4-P3), as well as their intermediate product, inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4), delayed increases in two new InsP4 isomers were consistently observed by high resolution high performance liquid chromatography. Studies on the metabolism of purified Ins-1,3,4,5-P4 preparations, labeled with [3H]inositol and 32P to monitor sites of dephosphorylation, were performed in permeabilized glomerulosa cells. In addition to rapid degradation of Ins-1,3,4,5-P3 to Ins-1,3,4-P3 and then to Ins-3,4-P2, there was delayed formation of one of the putative InsP4 isomers observed during AII stimulation in intact cells. The kinetics of formation of the new InsP4 isomer, and the lack of phosphate in its 5 position based on isotope ratios, were consistent with its origin from Ins-1,3,4-P3. This was confirmed by the conversion of [3H]Ins-1,3,4-P3 to the new InsP4 isomer in permeabilized cells by a kinase distinct from that which phosphorylates Ins-1,4,5-P3. These results have demonstrated that the dephosphorylation sequence of Ins-1,4,5-P3 metabolism is accompanied by a complex cycle of higher phosphorylations with formation of new intermediates of potential significance in cellular regulation.

Calretinin is a calcium-binding protein of the EF-hand family. It has been previously identified in particular cell types of adult guinea pig, rat, and chinchilla inner ear. Development of calretinin immunoreactivity in the mouse inner ear was investigated from embryonic day 13 (E13) to the adult stage. In the adult mouse vestibule, calretinin immunoreactivity was present in the same structures as described for the rat and guinea pig: the population of afferent fibers forming calyx units and a small number of ganglion neurons. The earliest immunoreactivity was found at E17 in vestibular hair cells (VHCs), then, at E19, in afferent fibers entering the sensory epithelia and in rare ganglion neurons. At postnatal day 4 (P4), a few vestibular nerve fibers and ganglion neurons were reactive. From this stage until P14, immunoreactivity developed in the calyx units and disappeared from VHCs. At P14, immunostaining was adult-like. In the adult mouse cochlea, immunoreactivity was present in the same cell populations as described in the rat: the inner hair cells (IHCs) and most of Corti's ganglion neurons. Calretinin immunoreactivity appeared at E19-P0 in IHCs and ganglion neurons of the basal turn. At P1, outer hair cells (OHCs) of the basal turn were positive. Calretinin immunoreactivity then appeared in IHCs, OHCs, and ganglion neurons of the medial turn, then of the apical turn. At P4, all IHCs and OHCs and most of the ganglion neurons were immunostained. Immunoreactivity gradually disappeared from the OHCs starting at P10 and, at P22, only IHCs and ganglion neurons were positive. The sequences of appearance of calretinin were specific to each cell type of the inner ear and paralleled their respective maturation. Calretinin was transiently expressed in VHCs and OHCs.

The carboxy terminus-encoding portion of the gag gene of Mason-Pfizer monkey virus (M-PMV), the prototype immunosuppressive primate type D retrovirus, encodes a 36-amino-acid, proline-rich protein domain that, in the mature virion, becomes the p4 capsid protein. The p4 domain has no known role in M-PMV replication. We found that two mutants with premature termination codons that remove half or all of the p4 domain produced lower levels of stable Gag protein and of self-assembled capsids. Interestingly, yeast two-hybrid screening revealed that p4 specifically interacted with TCP-1gamma, a subunit of the chaperonin TRiC (TCP-1 ring complex). TRiC is a cytosolic chaperonin that is known to be involved in both folding and subunit assembly of a variety of cellular proteins. TCP-1gamma also associated with high specificity with the M-PMV pp24/16-p12 domain and human immunodeficiency virus p6. Moreover, in cells, Gag polyprotein associated with the TRiC chaperonin complex and this association depended on ATP hydrolysis. In the p4 truncation mutants, the Gag-TRiC association was significantly reduced. These results strongly suggest that cytosolic chaperonin TRiC is involved in Gag folding and/or capsid assembly. We propose that TRiC associates transiently with nascent M-PMV Gag molecules to assist in their folding. Consequently, properly folded Gag molecules carry out the intermolecular interactions involved in self-assembly of the immature capsid.

An RNA fold is the result of packing together two or more coaxial helical stacks. To date, four RNA folds have been determined at near-atomic resolution by X-ray crystallography: transfer RNA, the hammerhead ribozyme, the P4-P6 domain of the Tetrahymena group I intron, and the hepatitis delta virus ribozyme. All four folds result in RNAs that are considerably more compact than isolated A-form duplexes. These structures illustrate, to varying degrees, three modes of fold stabilization: association of complementary molecular surfaces, stabilization of close RNA packing by binding of cations, and stabilization through pseudoknotting.

Low frequency (< 1 Hz) delta EEG in sleep is of increasing interest as it indicates cortical reorganization, especially in the prefrontal cortex (PFC). Other research shows that delta power in sleep is positively linked to waking cerebral metabolic rate. Such findings suggest that < 1 Hz activity may reflect waking performance at neuropsychological tests specific to the PFC. We investigated this unexplored area. Sleep EEGs (Fp1-F3, Fp2-F4, O1-P3, O2-P4) were recorded in 24 healthy 61-75-year-olds. We found significant associations between 0.5-1.0 Hz power from the left frontal EEG channel, in the first non-REM period, and performance at tasks more specific to the left PFC (e.g., nonverbal planning and verbal fluency). This association was absent from the posterior channels. Neither age nor response times were confounding factors. This potential sleep EEG marker for PFC neuropsychological function in healthy, older people also points to further uses of the sleep EEG in understanding the role of sleep.

The Kid and Kis proteins are the killer component and the antagonist belonging to parD, a killer stability system of plasmid R1. The Kid and Kis proteins have been purified, the second one as a C-LYT-Kis fusion that conserves the antagonistic activity of the Kis protein, but not its auto-regulatory potential. Kid inhibits in vitro replication of CoEl to a basal level without altering the superhelicity of the template but it does not substantially affect in vitro replication of P4, a DnaA, DnaB, DnaC and DnaG-independent replicon. Kid inhibits lytic induction of a lambda, prophage, but this inhibition can be neutralized by excess DnaB. In addition, a multicopy dnaB recombinant, but not a multicopy dnaG recombinant, prevents the toxicity associated with this protein. Inhibition of ColE1 replication by Kid in vitro is prevented by the C-LYT-Kis protein. Functional analysis indicates that the antagonistic activity of Kis is independent of its activity as a co-regulator of the parD promoter. It is also shown that C-LYT-Kis and Kid interact, forming a tight complex. These results strongly suggest that the toxicity of the kid protein is due to inhibition of DnaB-dependent DNA replication, and that direct protein-protein interactions are involved in the neutralization of the activity of the killer protein by the antagonist.

BACKGROUND

The human IGF2-P4 and H19 promoters are highly active in a variety of human cancers (including bladder cancer), while existing at a nearly undetectable level in the surrounding normal tissue.Single promoter vectors expressing diphtheria toxin A-fragment (DTA) under the control regulation of IGF2-P4 or H19 regulatory sequences (IGF2-P4-DTA and H19-DTA) were previously successfully used in cell lines, animal models and recently in human patients with superficial cell carcinoma of the bladder (treated with H19-DTA). However this targeted medicine approach could be limited, as not all cancer patients express high levels of H19. Hence, a double promoter DTA-expressing vector was created, carrying on a single construct two separate genes expressing the diphtheria toxin A-fragment (DTA), from two different regulatory sequences, selected from the cancer-specific promoters H19 and IGF2-P4.

METHODS

H19 and IGF2-P4 gene expression was tested in samples of Transitional Cell Carcinoma (TCC) of the bladder by in-situ hybridization (ISH) and by quantitative Real-Time PCR (qRT-PCR). The therapeutic potential of the double promoter toxin vector H19-DTA-IGF2-P4-DTA was tested in TCC cell lines and in heterotopic and orthotopic animal models of bladder cancer.

RESULTS

Nearly 100% of TCC patients highly expressed IGF2-P4 and H19, as determined by ISH and by qRT-PCR. The double promoter vector exhibited superior tumor growth inhibition activity compared to the single promoter expression vectors, in cell lines and in heterotopic and orthotopic bladder tumors.

CONCLUSIONS

Our findings show that bladder tumors may be successfully treated by intravesical instillation of the double promoter vector H19-DTA-P4-DTA.Overall, the double promoter vector exhibited enhanced anti-cancer activity relative to single promoter expression vectors carrying either gene alone.

Expression of a 4 kb segment of the bacteriocinogenic plasmid, pIP4P4, P5). With the exception of P4, which bears some resemblance to the consensus eubacterial promoter sequence, none of these promoters was recognized in vitro by the major forms of RNA polymerase from C. perfringens, Bacillus subtilis or Escherichia coli. Promoters P1, P3 and P5, which show striking homology with each other, contain unusual sequences in the '-35' and '-10' regions known to be recognized by RNA polymerase and this might indicate positive control.

Host cell RNA polymerase II-mediated transcription is inhibited by poliovirus infection. We have shown previously that the human TATA-binding protein (TBP), a general transcription factor required for transcription of all RNA polymerase II genes, is directly cleaved both in vitro and in vivo by the virus-coded protease 3CPro. 3CPro specifically cleaves glutamine-glycine bonds in the viral polyprotein. Cellular transcription factor TBP contains three glutamine-glycine sites, at amino acids 12, 18, and 108. By using site-directed mutagenesis, we determined that the glutamine-glycine bond at amino acid 18, but not that at amino acid 12 or 108, is cleaved by the viral protease. Both the glutamine and the glycine appear to be important for the cleavage. Further mutations around the glutamine-glycine site at position 18 suggest that determinants other than the glutamine-glycine bond in TBP are also required for 3CPro-induced cleavage. An alanine at position P4 and a proline at position P2, proximal to the scissile glutamine-glycine pair, appear to be important for 3CPro-mediated cleavage of TBP. Our results suggest that the cleavage specificity of 3CPro for a cellular transcription factor is very similar to its mode of cleavage of viral polyproteins.

Invasive carcinoma cells form actin-rich matrix-degrading protrusions called invadopodia. These structures resemble podosomes produced by some normal cells and play a crucial role in extracellular matrix remodeling. In cancer, formation of invadopodia is strongly associated with invasive potential. Although deregulated signals from the receptor tyrosine kinase Met (also known as hepatocyte growth factor are linked to cancer metastasis and poor prognosis, its role in invadopodia formation is not known. Here we show that stimulation of breast cancer cells with the ligand for Met, hepatocyte growth factor, promotes invadopodia formation, and in aggressive gastric tumor cells where Met is amplified, invadopodia formation is dependent on Met activity. Using both GRB2-associated-binding protein 1 (Gab1)-null fibroblasts and specific knockdown of Gab1 in tumor cells we show that Met-mediated invadopodia formation and cell invasion requires the scaffold protein Gab1. By a structure-function approach, we demonstrate that two proline-rich motifs (P4/5) within Gab1 are essential for invadopodia formation. We identify the actin regulatory protein, cortactin, as a direct interaction partner for Gab1 and show that a Gab1-cortactin interaction is dependent on the SH3 domain of cortactin and the integrity of the P4/5 region of Gab1. Both cortactin and Gab1 localize to invadopodia rosettes in Met-transformed cells and the specific uncoupling of cortactin from Gab1 abrogates invadopodia biogenesis and cell invasion downstream from the Met receptor tyrosine kinase. Met localizes to invadopodia along with cortactin and promotes phosphorylation of cortactin. These findings provide insights into the molecular mechanisms of invadopodia formation and identify Gab1 as a scaffold protein involved in this process.

HIV-1 develops resistance to protease inhibitors predominantly by selecting mutations in the protease gene. Studies of resistant mutants of HIV-1 protease with single amino acid substitutions have shown a range of independent effects on specificity, inhibition, and stability. Four double mutants, K45I/L90M, K45I/V82S, D30N/V82S, and N88D/L90M were selected for analysis on the basis of observations of increased or decreased stability or enzymatic activity for the respective single mutants. The double mutants were assayed for catalysis, inhibition, and stability. Crystal structures were analyzed for the double mutants at resolutions of 2.2-1.2 A to determine the associated molecular changes. Sequence-dependent changes in protease-inhibitor interactions were observed in the crystal structures. Mutations D30N, K45I, and V82S showed altered interactions with inhibitor residues at P2/P2', P3/P3'/P4/P4', and P1/P1', respectively. One of the conformations of Met90 in K45I/L90M has an unfavorably close contact with the carbonyl oxygen of Asp25, as observed previously in the L90M single mutant. The observed catalytic efficiency and inhibition for the double mutants depended on the specific substrate or inhibitor. In particular, large variation in cleavage of p6(pol)-PR substrate was observed, which is likely to result in defects in the maturation of the protease from the Gag-Pol precursor and hence viral replication. Three of the double mutants showed values for stability that were intermediate between the values observed for the respective single mutants. D30N/V82S mutant showed lower stability than either of the two individual mutations, which is possibly due to concerted changes in the central P2-P2' and S2-S2' sites. The complex effects of combining mutations are discussed.

Factor (f) IXa is a critical enzyme for the formation of stable blood clots, and its deficiency results in hemophilia. The enzyme functions at the confluence of the intrinsic and extrinsic pathways by binding to fVIIIa and rapidly generating fXa. In spite of its importance, little is known about how fIXa recognizes its cofactor, its substrate, or its only known inhibitor, antithrombin (AT). However, it is clear that fIXa requires extensive exosite interactions to present substrates for efficient cleavage. Here we describe the 1.7-A crystal structure of fIXa in its recognition (Michaelis) complex with heparin-activated AT. It represents the highest resolution structure of both proteins and allows us to address several outstanding issues. The structure reveals why the heparin-induced conformational change in AT is required to permit simultaneous active-site and exosite interactions with fIXa and the nature of these interactions. The reactive center loop of AT has evolved to specifically inhibit fIXa, with a P2 Gly so as not to clash with Tyr99 on fIXa, a P4 Ile to fit snugly into the S4 pocket, and a C-terminal extension to exploit a unique wall-like feature of the active-site cleft. Arg150 is at the center of the exosite interface, interacting with AT residues on beta-sheet C. A surprising crystal contact is observed between the heparin pentasaccharide and fIXa, revealing a plausible mode of binding that would allow longer heparin chains to bridge the complex.

A new set of bis(5'-nucleosidyl) tetraphosphates, the Bp4B' nucleotides (B and B' = C, G, or U not equal to A), are demonstrated in living cells. In exponentially growing Saccharomyces cerevisiae, cellular concentrations of Cp4U, Up4U, Gp4G, Cp4C, Gp4U, and Gp4C are 210, 200, 60, 50, 40, and 30 nM, respectively. It is likely that these nucleotides originate from the action of diadenosine-5',5"'-P1,P4-tetraphosphate alpha,beta-phosphorylase, an enzyme recently found in yeast. Upon temperature shift or exposure to cadmium, the Bp4B' nucleotides strongly accumulate in the yeast cells. In Escherichia coli, the same nucleotides occur, and similar effects of temperature shift or of cadmium are observed. However, in the bacterium, the origin of these nucleotides is not known. To quantitate these nucleotides in cellular extracts, specific procedures were developed. In the first step, after purification of the mixture of Np4N' (N and N' = A, C, G, or U) nucleotides, the Ap4N nucleotides are specifically removed by incubation with lysyl-tRNA synthetase. In the second step, the Bp4B' species are resolved with the help of anion-exchange high performance liquid chromatography. In the third step, the concentration of each Bp4B' is measured using three coupled enzymatic reactions to produce ATP and bioluminescence. With this strategy, 0.01 pmol of any Bp4B' nucleotide can be reliably detected.

P4-ATPases (flippases) translocate specific phospholipids such as phosphatidylserine from the exoplasmic leaflet of the cell membrane to the cytosolic leaflet, upholding an essential membrane asymmetry. The mechanism of flipping this giant substrate has remained an enigma. We have investigated the importance of amino acid residues in transmembrane segment M4 of mammalian P4-ATPase ATP8A2 by mutagenesis. In the related ion pumps Na(+),K(+)-ATPase and Ca(2+)-ATPase, M4 moves during the enzyme cycle, carrying along the ion bound to a glutamate. In ATP8A2, the corresponding residue is an isoleucine, which recently was found mutated in patients with cerebellar ataxia, mental retardation, and dysequilibrium syndrome. Our analyses of the lipid substrate concentration dependence of the overall and partial reactions of the enzyme cycle in mutants indicate that, during the transport across the membrane, the phosphatidylserine head group passes near isoleucine-364 (I364) and that I364 is critical to the release of the transported lipid into the cytosolic leaflet. Another M4 residue, N359, is involved in recognition of the lipid substrate on the exoplasmic side. Our functional studies are supported by structural homology modeling and molecular dynamics simulations, suggesting that I364 and adjacent hydrophobic residues function as a hydrophobic gate that separates the entry and exit sites of the lipid and directs sequential formation and annihilation of water-filled cavities, thereby enabling transport of the hydrophilic phospholipid head group in a groove outlined by the transmembrane segments M1, M2, M4, and M6, with the hydrocarbon chains following passively, still in the membrane lipid phase.

Cellular membranes, notably eukaryotic plasma membranes, are equipped with special proteins that actively translocate lipids from one leaflet to the other and thereby help generate membrane lipid asymmetry. Among these ATP-driven transporters, the P4 subfamily of P-type ATPases (P4-ATPases) comprises lipid flippases that catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of cell membranes. While initially characterized as aminophospholipid translocases, recent studies of individual P4-ATPase family members from fungi, plants, and animals show that P4-ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates, including lysophospholipids and synthetic alkylphospholipids. At the same time, the cellular processes known to be directly or indirectly affected by this class of transporters have expanded to include the regulation of membrane traffic, cytoskeletal dynamics, cell division, lipid metabolism, and lipid signaling. In this review, we will summarize the basic features of P4-ATPases and the physiological implications of their lipid transport activity in the cell.

Log-transformed EEG power spectral estimates (6-7, 9,10-11,18-22, and 36-44 Hz), obtained from skilled marksmen and novice shooters at sites F3, F4, C3, C4,T3, T4, P3, P4, O1, and O2 during the aiming period (6 s) of a target shooting task for each of 40 trials up to the moment of trigger pull, were contrasted to determine regional differences in cortical activation. The EEG power obtained from both groups during the preparatory aiming period was also compared to that observed for a similar time period during the processing of standard verbal and spatial tasks. The marksmen exhibited less activation than the novice shooters at all sites during the aiming period with a pronounced difference in the left central-temporal-parietal area. Fewer group differences in cortical activation were observed during the comparative verbal and spatial tasks with which the groups held equal experience. Additionally, the novice shooters exhibited a cortical activation pattern during target shooting that was similar to that observed during the processing of the comparative verbal and spatial tasks. In contrast, marksmen generally exhibited less cortical activation during the aiming period when contrasted to that during the novel comparative tasks. These results are consistent with the notion of relative economy in the cortical processes of marksmen, relative to controls, during the specific challenge with which they are highly practiced.

CONSPECTUS: Carbenes are compounds that feature a divalent carbon atom with only six electrons in its valence shell. In the singlet state, they possess a lone pair of electrons and a vacant orbital and therefore exhibit Lewis acidic and Lewis basic properties, which explains their very high reactivity. Following the preparation by our group in 1988 of the first representative, a variety of stable carbenes are now available, the most popular being the cyclic diaminocarbenes. In this Account, we discuss another class of stable cyclic carbenes, namely, cyclic (alkyl)(amino)carbenes (CAACs), in which one of the electronegative and π-donor amino substituents of diaminocarbenes is replaced by a σ-donating but not π-donating alkyl group. As a consequence, CAACs are more nucleophilic (σ-donating) but also more electrophilic (π-accepting) than diaminocarbenes. Additionally, the presence of a quaternary carbon in the position α to the carbene center provides steric environments that differentiate CAACs dramatically from all other ligands. We show that the peculiar electronic and steric properties of CAACs allow for the stabilization of unusual diamagnetic and paramagnetic main group element species. As examples, we describe the preparation of room temperature stable phosphorus derivatives in which the heteroatom is in the zero oxidation state, nucleophilic boron compounds, and phosphorus-, antimony-, boron-, silicon-, and even carbon-centered neutral and cationic radicals. CAACs are also excellent ligands for transition metal complexes. The most recent application is their use for the stabilization of paramagnetic complexes, in which the metal is often in a formal zero oxidation state. Indeed, bis(CAAC)M complexes in which the metal is gold, copper, cobalt, iron, nickel, manganese, and zinc have been isolated. Depending on the metal, the majority of spin density can reside either on the metal or on the carbene carbons and the nitrogen atoms of the CAAC ligand. In contrast to diaminocarbenes, the higher basicity of CAACs makes them poor leaving groups, and thus they cannot be used for classical organocatalysis. However, because of their superior electrophilicity and smaller singlet-triplet gap, CAACs can activate small molecules at room temperature, such as CO, H2, and P4, as well as enthalpically strong bonds, such as B-H, Si-H, N-H, and P-H. Lastly, excellent results have been obtained in palladium, ruthenium, and gold catalysis. CAAC-metal complexes are extremely thermally robust, which allows for their utilization in harsh conditions. This property has been used to perform a variety of gold-catalyzed reactions in the presence of basic amines, including ammonia and hydrazine, which usually deactivate catalysts.

BACKGROUND

Ethanol produces abnormalities in the developing nervous system, with certain regions being vulnerable during well-defined periods. Neonatal rodent cerebellum is particularly susceptible to ethanol during the early postnatal period [on postnatal days 4-5 (P4-5)], while this region is resistant to ethanol at a slightly later time (P7-9). We assessed basal levels of several substances which may be involved in differential temporal ethanol vulnerability in neonatal cerebellum, and analyzed alterations in these substances after early ethanol exposure.

METHODS

Assessments were made of neurotrophic factors nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4; apoptosis-related proteins Bcl-2, Bcl-xl, Bax, Bcl-xs, Bad, phosphorylated-Bad, phosphorylated-Akt, and phosphorylated-c-Jun N-terminal kinase; and the antioxidants superoxide dismutase, glutathione reductase, and catalase. These analyses quantified basal levels (in controls), and sequential changes following acute ethanol exposure at the vulnerable and resistant cerebellar periods (P4, P7).

RESULTS

Comparisons of basal levels of the molecules assessed between P4 and P7 revealed higher levels of total proapoptotic Bad at p4, higher levels of the protective pAkt kinase at P7, and lower levels of proapoptotic pJNK at P7. Other basal levels did not differ. While ethanol-mediated alterations were found at both ages favoring both apoptosis and survival, the apoptosis-promoting changes produced on P4 exceeded those on P7, and most occurred within the first 2 hr after exposure, a critical survival/death period. The number of alterations favoring survival were similar at the two ages, but at P7 most occurred within the first 2 hr after exposure, possibly acting in a protective manner.

CONCLUSIONS

Differential temporal vulnerability to ethanol in the neonatal cerebellum appears to be paralleled by cellular alterations in neurotrophic factors, apoptosis-regulatory proteins, and/or antioxidant activities which generally favor apoptosis at the most sensitive age and survival at the resistant age.

Insects have an efficient defense system against infections. Their antibacterial immune proteins have been well characterized. However, the molecular mechanisms by which insects recognize foreignness are not yet known. Data are presented showing that hemolin (previously named P4), a bacteria-inducible hemolymph protein of the giant silk moth Hyalophora cecropia, belongs to the immunoglobulin superfamily. Functional analyses indicate that hemolin is one of the first hemolymph components to bind to the bacterial surface, taking part in a protein complex formation that is likely to initiate the immune response.

Insulin-like growth factor-2 (IGF-2) is expressed in most embryonic tissues and is required for normal development during gestation. After birth IGF-2 expression is extinguished in most tissues, but the gene is often reactivated during tumorigenesis. Tumors secrete high molecular weight forms of IGF-2 that result from aberrant post-translational processing of pro-IGF-2. As a first step toward understanding how high molecular weight IGF-2 peptides might contribute to tumor progression, we have characterized the biosynthesis of IGF-2 in a human embryonic cell line. We have found that pro-IGF-2 can initially form two disulfide isomers that undergo rearrangement to a single conformation in vivo. The addition of N-acetylgalactosamine to Ser71, Thr72, Thr75, and Thr139 likely occurs in the cis- Golgi apparatus. Sialic acid addition begins in the trans- Golgi apparatus, but IGF-2 peptides must reach the trans-Golgi network for oligosaccharide maturation to be completed. Endoproteolysis occurs concomitant to or slightly after oligosaccharide maturation. Cleavage was observed only at Arg104, resulting in the secretion of IGF-2-(1-104) and free E-peptide. Proteolysis required basic residues in the P1 (Arg104) and P4 (Arg101) positions, was completely blocked by a furin inhibitor, and was enhanced by coexpression with furin, PACE4, PC6A, PC6B, and LPC. These data suggest that members of the subtilisin-related proprotein convertase family mediate processing of pro-IGF-2 at Arg104. We did not detect the IGF-2 peptides that are most abundant in normal serum, mature IGF-2, and IGF-2-(1-87), in this expression system, which indicates that novel endoproteases are responsible for generating these products.

In the mouse, estrogen is essential for blastocyst implantation in the progesterone (P4)-primed uterus. The mechanism(s) by which estrogen initiates this response still remains elusive. The present investigation, using delayed implantation in the mouse, examined the differential role of estradiol-17beta (E2) and its catechol metabolite 4-hydroxy-E2 (4-OH-E2) in uterine and blastocyst activation for implantation. The conditions of delayed implantation were induced by ovariectomizing mice on day 4 (day 1 = vaginal plug) of pregnancy or pseudopregnancy and maintaining them with P4 from days 5-7. The binding of EGF to blastocysts was used as a marker for blastocyst activation. Our results show that whereas E2 fails to activate dormant blastocysts (with respect to EGF binding in vitro), 4-OH-E2, cAMP, or prostaglandin E2, is effective in this response. Further, whereas 4-OH-E2 induced-activation is not blocked by an antiestrogen, an inhibitor of PG synthesis, adenylyl cyclase or protein kinase A effectively blocks this activation. These results suggest that 4-OH-E2 effects on blastocysts are mediated by PGs, which, in turn, stimulate cAMP production and thus activation of protein kinase A. Two-fluoro-E2 is a poor substrate and an inhibitor of catecholestrogen synthesis, but it is estrogenic, with respect to uterine growth and gene expression. Using blastocyst transfer experiments, we observed that dormant blastocysts incubated with 4-OH-E2 in vitro, but not with E2, are capable of implanting in P4-treated delayed implanting mice receiving two-fluoro-E2. The results suggest that whereas E2 is necessary for preparation of the uterus, uterine-derived catecholestrogen is important for blastocyst activation for implantation. Indeed, the receptive uterus has the capacity to synthesize 4-OH-E2. Collectively, we demonstrate that the primary ovarian estrogen E2, via its interaction with nuclear estrogen receptors, participates in the preparation of the P4-primed uterus to the receptive state in an endocrine manner, whereas its metabolite 4-OH-E2, produced from E2 in the uterus, mediates blastocyst activation for implantation in a paracrine manner. Our results also establish that these target-specific effects of primary estrogen and catecholestrogen are both essential for implantation and that successful implantation occurs only when the activated stage of the blastocyst coincides with the receptive state of the uterus.

The dinucleotide AppppA (5',5'''-P1, P4-diadenosine tetraphosphate) is rapidly synthesized in cells exposed to heat stress or oxidative stress. Stress-induced AppppA accumulation has been observed in all cell types studied to date. In order to study the function(s) of AppppA, we created a mutation in the Escherichia coli gene that encodes the sole AppppN hydrolase (apaH). High levels of AppppA have subsequently been shown to affect many cellular processes, including expression of catabolite repressible genes and the ability to survive starvation, oxidative stress and near-UV irradiation. Nevertheless, the precise role of AppppA remains undefined. In order to better understand the mechanism by which AppppA exerts its effects, we attempted to determine which proteins bind to AppppA by synthesizing (alpha'-32P) 8-N3AppppA for use in photocrosslinking experiments with extract derived from cells with different genetic backgrounds and exposed to various stress conditions. We report here that several E. coli proteins bind AppppA, including the heat shock and oxidative stress proteins DnaK, GroEL, E89, C45 and C40. In addition, we show that apaH mutants, which have high basal levels of AppppA, are hypersensitive to killing by heat.

Progesterone (P4) has emerged as an important hormone-regulating mammary stem cell (MaSC) populations. In breast cancer, P4 and synthetic analogs increase the number of stem-like cells within luminal estrogen receptor (ER)- and progesterone receptor (PR)-positive breast cancers. These cells gain expression of de-differentiated cell markers CD44 and cytokeratin 5 (CK5), lose luminal markers ER and PR, and are more therapy resistant. We previously described that P4 downregulation of microRNA (miR)-29a contributes to the expansion of CD44(high) and CK5(+) cells. Here we investigated P4 downregulation of miR-141, a member of the miR-200 family of tumor suppressors, in facilitating an increase in stem-like breast cancer cells. miR-141 was the sole member of the miR-200 family P4-downregulated at the mature miRNA level in luminal breast cancer cell lines. Stable inhibition of miR-141 alone increased the CD44(high) population, and potentiated P4-mediated increases in both CD44(high) and CK5(+) cells. Loss of miR-141 enhanced both mammosphere formation and tumor initiation. miR-141 directly targeted both PR and signal transducer and activator of transcription 5A (Stat5a), transcription factors important for MaSC expansion. miR-141 depletion increased PR protein levels, even in cell lines where PR expression is estrogen dependent. Stat5a suppression via small interfering RNA or a small-molecule inhibitor reduced the P4-dependent increase in CK5(+) and CD44(high) cells. These data support a mechanism by which P4-triggered loss of miR-141 facilitates breast cancer cell de-differentiation through deregulation of PR and Stat5a, two transcription factors important for controlling mammary cell fate.