Somatosensory thalamus and cortex in rodents contain topological representations of the facial whisker pad. The thalamic representation of a single whisker ("barreloid") is presumed to project exclusively to the cortical representation ("barrel") of the same whisker; however, it was not known when this correspondence is established during early development, nor how precise the thalamocortical projection is at birth, before formation of barrels and barreloids. To answer these questions, we retrogradely labeled thalamocortical projection neurons in fixed brain slices from 0-8 d old (P0-P8) mice, by placing paired deposits of two fluorescent dyes in adjacent barrels or (before barrel formation) in adjacent loci in upper cortical layers. At all ages studied, a negligible fraction of the retrogradely labeled cells was double labeled, implying that branches of single thalamocortical axons never extended within layer IV over an area wider than a single barrel. In P0 preparations, 70% of paired dye deposits placed 75-200 microns apart resulted in statistically significant segregation of labeled cell clusters in the thalamus. Quantitative analysis indicated that on P0 about 70% of thalamocortical axons were within 1.3 presumptive barrel diameters from their topologically precise target. In P4-P8 preparations, the great majority of thalamic cells retrogradely labeled from a single barrel were found in a single barreloid, implying a 1:1 projection of barreloids to barrels. The postnatal increase in topological precision was reproduced by a computer simulation, which assumed that many aberrant axons corrected their initial targeting error by extending terminal arborizations asymmetrically, towards the center of their appropriate barrel.

In the generation of flavivirus particles, an internal cleavage of the envelope glycoprotein prM by furin is required for the acquisition of infectivity. Unlike cleavage of the prM of other flaviviruses, cleavage of dengue virus prM is incomplete in many cell lines; the partial cleavage reflects the influence of residues at furin nonconsensus positions of the pr-M junction, as flaviviruses share basic residues at positions P1, P2, and P4, recognized by furin. In this study, viruses harboring the alanine-scanning and other multiple-point mutations of the pr-M junction were generated, employing a dengue virus background that exhibited 60 to 70% prM cleavage and a preponderance of virion-sized extracellular particles. Analysis of prM and its cleavage products in viable mutants revealed a cleavage-suppressive effect at the conserved P3 Glu residue, as well as the cleavage-augmenting effects at the P5 Arg and P6 His residues, indicating an interplay between opposing modulatory influences mediated by these residues on the cleavage of the pr-M junction. Changes in the prM cleavage level were associated with altered proportions of extracellular virions and subviral particles; mutants with reduced cleavage were enriched with subviral particles and prM-containing virions, whereas the mutant with enhanced cleavage was deprived of these particles. Alterations of virus multiplication were detected in mutants with reduced prM cleavage and were correlated with their low specific infectivities. These findings define the functional roles of charged residues located adjacent to the furin consensus sequence in the cleavage of dengue virus prM and provide plausible mechanisms by which the reduction in the pr-M junction cleavability may affect virus replication.

A recently reported crystal structure of an intact bacterial group I self-splicing intron in complex with both its exons provided the first molecular view into the mechanism of RNA splicing. This intron structure, which was trapped in the state prior to the exon ligation reaction, also reveals the architecture of a complex RNA fold. The majority of the intron is contained within three internally stacked, but sequence discontinuous, helical domains. Here the tertiary hydrogen bonding and stacking interactions between the domains, and the single-stranded joiner segments that bridge between them, are fully described. Features of the structure include: (1) A pseudoknot belt that circumscribes the molecule at its longitudinal midpoint; (2) two tetraloop-tetraloop receptor motifs at the peripheral edges of the structure; (3) an extensive minor groove triplex between the paired and joiner segments, P6-J6/6a and P3-J3/4, which provides the major interaction interface between the intron's two primary domains (P4-P6 and P3-P9.0); (4) a six-nucleotide J8/7 single stranded element that adopts a mu-shaped structure and twists through the active site, making critical contacts to all three helical domains; and (5) an extensive base stacking architecture that realizes 90% of all possible stacking interactions. The intron structure was validated by hydroxyl radical footprinting, where strong correlation was observed between experimental and predicted solvent accessibility. Models of the pre-first and pre-second steps of intron splicing are proposed with full-sized tRNA exons. They suggest that the tRNA undergoes substantial angular motion relative to the intron between the two steps of splicing.

Foamy viruses (FVs) express the Gag protein as a precursor with a molecular mass of 74 kDa (pr74) from which a 70-kDa protein (p70) is cleaved by the viral protease. To gain a better understanding of FV Gag protein processing and function, we have generated and analyzed mutants in the C-terminal gag region of an infectious molecular clone. Our results show that p70 is an N-terminal cleavage product of pr74. However, we were unable to identify a p4 molecule. A virus mutant expressing p70 only was found to be replication competent, albeit at very low titers compared to those of wild-type virus. A strong tendency to synthesize and cleave a pr74 molecule was deduced from the occurrence of revertants upon transfection of this mutant. Substitution of the p6gag domain of human immunodeficiency virus type 1 for the p4 domain of FV resulted in a stable chimeric virus which replicated to titers 10 times lower than those of wild-type virus. FV Gag protein was found to be phosphorylated at serine residues. Mutagenesis of serines conserved in the p4 domain had no influence on viral replication in cell culture. The p70/p74 Gag cleavage was found to be required for viral infectivity, since mutagenesis of the putative cleavage site led to replication-incompetent virus. Interestingly, the cleavage site mutants were defective in the intracellular cDNA synthesis of virion DNA, which indicates that correct FV particle formation and the generation of virion DNA are functionally linked.

Mechanisms maintaining the growth of a "semi-foreign" fetus within the maternal uterus via immune tolerance remain unclear. CD4(+)CD25(+) regulatory T (Treg) cells have been implicated in the maintenance of maternal-fetal immune tolerance. Additionally, 17β-estradiol (E2) is able to initiate immune suppression through CD4(+)CD25(+) Treg cells during early pregnancy. Little is known, however, regarding the relationship between progesterone (P4) and immune tolerance during midterm pregnancy, an important period, characterized by higher levels of P4 but lower levels of E2 in the serum. Here, we examined the effects of P4 on the expansion and function of systemic and local uterine CD4(+)CD25(+) Treg cells during midterm pregnancy in mice. Using in vivo and in vitro models, we provide the first evidence that P4 not only increases the proportion of CD4(+)CD25(+) Treg cells and IL-10 expression but also enhances their suppressive function. Moreover, at physiological doses relevant to midterm pregnancy, P4, but not E2, converts CD4(+)CD25(-) T cells into CD4(+)CD25(+) Treg cells. This conversion was inhibited in vitro by the nuclear P4 receptors antagonist RU 486 and in vivo in P4-treated ovariectomized and pseudopregnant mice models, suggesting that P4 expands Treg populations via nuclear P4 receptors. Furthermore, RU 486 significantly reduced the quantity and function of Treg cells in the fetal-maternal interface before the onset of induced abortion. Interestingly, with decreasing Foxp3, proinflammatory factors increased. Together, the present results demonstrate that P4 is an important regulator of systemic and local CD4(+)CD25(+) Treg cells, which are involved in maintaining maternal-fetal immune tolerance during midterm pregnancy.

The proprotein processing activity of mutants of the subtilisin-like enzyme furin was studied in transfected mammalian cells. Our studies indicate that the three residues of the catalytic triad of furin, Asp46, His87, and Ser261, are critical not only for substrate processing but also for maturation of furin. Furthermore, evidence is provided that maturation of furin occurs through an intramolecular autocatalytic process. Substitution of the asparagine residue (Asn188) of the oxyanion hole by an alanine residue appears to block substrate processing but not furin maturation. Analysis of carboxyl-terminal deletion mutants revealed that the segment encompassing residues Glu449 to Glu469 of the "middle" domain, which is more than 100 residues downstream of the predicted catalytic domain, contains residues that seem to be critical for processing activity but that the more carboxyl-terminal cysteine-rich region, the transmembrane region, and the cytosolic tail are dispensable. Finally, we made mutants in the substrate binding region of human furin and studied their ability to process von Willebrand factor (pro-vWF) substrates, including wild-type pro-vWF as well as pro-vWF mutants in which the P1 (vWFR-1G), P2 (vWFK-2A), or P4 (vWFR-4A) basic residue with respect to the pro region cleavage site had been mutated. It is demonstrated that particular negatively charged residues in or near the substrate binding region of furin are critical for cleavage activity and specificity of the enzyme for multiple basic residues in the substrate. Furthermore, substrate binding region mutants of furin were obtained, which cleaved either the pro-vWFK-2A or pro-vWFR-4A mutant of pro-vWF more efficiently than wild-type pro-vWF.

The three-dimensional structure of the calcium-free form of Bacillus licheniformis alpha-amylase (BLA) has been determined by multiple isomorphous replacement in a crystal of space group P4(3)2(1)2 (a = b = 119.6 A, c = 85.4 A). The structure was refined using restrained crystallographic refinement to an R-factor of 0.177 for 28,147 independent reflections with intensities FObs>> 0 at 2.2 A resolution, with root mean square deviations of 0.008 A and 1.4 degrees from ideal bond lengths and bond angles, respectively. The final model contains 469 residue, 237 water molecules, and one chloride ion. The segment between Trp182 and Asn192 could not be located in the electron density, nor could the N and C termini. Cleavage of the calcium-free form of BLA was observed after Glu189, due to a Glu-C endopeptidase present in trace amounts in the preparation. BLA did not crystallize without this cleavage under the conditions applied. BLA exhibits the characteristic overall topological fold observed for other alpha-amylases and related amylolytic enzymes: a central domain A containing an alpha/beta-barrel with a large protrusion between beta-strand 3 and alpha-helix 3 (domain B) and a C-terminal greek key motif (domain C). Unlike in the other enzymes, domain B possesses a beta-sheet made up of six loosely connected, twisted beta-strands forming a kind of a barrel with a large hole in the interior. Topological comparisons to TAKA-amylase, pig pancreatic alpha-amylase and cyclodextrin glycosyltransferase reveal a very high structural equivalence for large portions of the proteins and an exceptionally pronounced structural similarity for calcium binding, chloride binding and the active site. None of the theories proposed to explain the enhanced thermostability of BLA showed a satisfactory correlation with the three-dimensional structure. Instead, sequence comparisons to the less thermostable bacterial alpha-amylase from Bacillus amyloliquefaciens (BAA) indicate that some ionic interactions present in BLA, but which cannot be formed in BAA, might be responsible for the enhanced thermostability of BLA.

The cis elements involved in the cAMP regulation of transcription of the gene for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) were studied by introducing a series of block mutations (10-15 base pairs of random sequence) into eight of the protein binding domains in a region of the promoter between -490 and +73. Each mutant promoter was ligated to the structural gene for chloramphenicol acetyltransferase (CAT) and transfected into HepG2 cells. Transcription of PEPCK-CAT was stimulated 4-fold by the addition of 8-bromo-cAMP (8-Br-cAMP), whereas overexpression of the catalytic subunit of protein kinase A in these cells increased transcription from the PEPCK promoter 30-fold. Several elements within the PEPCK promoter acted synergistically to mediate this effect. These include CRE-1 (-92 to -82) and a complex unit from -220 to -280 composed of multiple binding sites termed P3(I) (-250 to -234), P3(II) (-260 to -250), and P4 (-286 to -270). Mutation of both CRE-1 and P3(I) resulted in the complete elimination of transcriptional induction by either 8-Br-cAMP or the catalytic subunit of protein kinase A. To examine the proteins involved in this response, we replaced CRE-1, which binds both C/EBP and cAMP-responsive element binding protein (CREB), with an optimal C/EBP binding sequence which significantly decreased the binding of CREB, but maintained the affinity for C/EBP. Transcription from this modified promoter was induced by 8-Br-cAMP and the catalytic subunit of protein kinase A (PKA) to a similar extent as noted with the native PEPCK promoter. However, the results of experiments involving cotransfection of PEPCK-CAT with expression vectors for PKA and either C/EBP or CREB suggest that CREB is capable of mediating a greater responsiveness to PKA than C/EBP. Our results indicate that multiple cis elements are involved in the cAMP induction of PEPCK gene transcription and that C/EBP and CREB are potentially involved in this response.

The role of Ca2+ in the generation of inositol phosphates was investigated using rat pancreatic islets after steady state labeling with myo-[2-3H]inositol. Depolarizing K+ concentrations (24 mM) evoked early (2 s) increases in inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P4) as measured by high performance anion-exchange chromatography. The increase in Ins-1,4,5-P3 was transient and was followed by a more pronounced rise in Ins-1,3,4-P3. These effects were dependent on the presence of extracellular Ca2+ but were not secondary to release of either neurotransmitters or metabolites of arachidonic acid. K+ also promoted the breakdown of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) and of the other phosphoinositides. Glucose (16.7 mM) was less marked in its effects but still promoted rapid increases in Ins-1,3,4,5-P4 (2 s) and Ins-1,4,5-P3 (10 s) and a slower rise in Ins-1,3,4-P3 (30 s). The levels of all three metabolites rose steadily over 10 min stimulation. These responses to glucose could be largely, although not entirely, inhibited by depletion of extracellular Ca2+ or by Ca2+ channel blockade with verapamil (20 microM). Carbamylcholine (0.5 mM) was the most potent stimulus used evoking early rises in Ins-1,4,5-P3 and Ins-1,3,4,5-P4 (2 s) followed by Ins-1,3,4-P3 (10 s), effects which were only partially dependent on extracellular Ca2+. The results suggest that a Ca2+-mediated PtdIns-4,5-P2 hydrolysis accounts for most of the Ins-1,4,5-P3 generated in response to glucose but not carbamylcholine. In addition, glucose may exert effects on inositol phosphate metabolism which are Ca2+ independent.

Herein, we show that a single injection of P4 (4 mg/kg) at 1 h or 48 h, but not 96 h, before middle cerebral artery occlusion (MCAO) produces significant protective effects against the ischemia-induced neuronal death and the deficits in spatial cognition and LTP induction. The present study focused on the molecular mechanisms underlying the neuroprotection exerted by P4 administration at 1 h and 48 h pre-MCAO, termed acute and delayed P4-neuroprotection, respectively. Pharmacology suggested that P4-receptor (P4R) cascading to a Src-ERK1/2 signaling mediated the delayed P4-neuroprotection. To support this, it was observed by anti-phosph-ERK1/2 immunoblots that a single injection of P4 triggered a P4R-mediated persistent increase in ERK1/2 phosphorylation and their nuclear translocation for 48 h. In contrast, the acute P4-neuroprotection did not depend on the P4R-mediated Src-ERK1/2 signaling. Instead, the acute P4-administration attenuated the NMDA-induced rise in the intracellular calcium concentration ([Ca(2+)](i)) that may be a primary cause for MCAO-induced neuronal injury. This effect seemed to be exerted by an antagonism of sigma(1) receptor since the sigma(1) receptor antagonist NE100 perfectly mimicked the acute P4-neuroprotection and also attenuated the NMDA-induced [Ca(2+)](i) increase. These findings suggest that the P4 neuroprotection involves two independent processes depending on the timing of P4 administration before MCAO: an acute protection by antagonizing sigma(1) receptor to inhibit NMDAr-Ca(2+) influx and a delayed one by an activation of P4R-mediated Src-ERK signaling pathway.

The pre-Bötzinger complex is a small region in the mammalian brainstem involved in generation of the respiratory rhythm. As shown in vitro, this region, under certain conditions, can generate endogenous rhythmic bursting activity. Our investigation focused on the conditions that may induce this bursting behaviour. A computational model of a population of pacemaker neurons in the pre-Bötzinger complex was developed and analysed. Each neuron was modelled in the Hodgkin-Huxley style and included persistent sodium and delayed-rectifier potassium currents. We found that the firing behaviour of the model strongly depended on the expression of these currents. Specifically, bursting in the model could be induced by a suppression of delayed-rectifier potassium current (either directly or via an increase in extracellular potassium concentration, [K+]o) or by an augmentation of persistent sodium current. To test our modelling predictions, we recorded endogenous population activity of the pre-Bötzinger complex and activity of the hypoglossal (XII) nerve from in vitro transverse brainstem slices (700 micro m) of neonatal rats (P0-P4). Rhythmic activity was absent at 3 mm[K+]o but could be triggered by either the elevation of [K+]o to 5-7 mm or application of potassium current blockers (4-AP, 50-200 micro m, or TEA, 2 or 4 mm), or by blocking aerobic metabolism with NaCN (2 mm). This rhythmic activity could be abolished by the persistent sodium current blocker riluzole (25 or 50 micro m). These findings are discussed in the context of the role of endogenous bursting activity in the respiratory rhythm generation in vivo vs. in vitro and during normal breathing in vivo vs. gasping.

The epithelial and stromal compartments of the uterus undergo significant estrogen- and progesterone (P4)-induced changes during the estrous cycle. While in the adult mouse, epithelial proliferation and stromal inflammation are induced by estrogen, P4 is antiproliferative in the epithelium and both proliferative and anti-inflammatory in the stroma. In light of these compartmentally varying roles, we have immunohistochemically examined estrogen and P4 regulation of the expression of their receptors (ER and PR) and their epithelial target gene lactoferrin (LF) in wild-type and PR null mutant mice. We demonstrate that estrogen exerts compartment-specific effects on the expression of ER, resulting in decreased levels of stromal and glandular epithelial (GE) ER and increased luminal epithelial (LE) and myometrial ER. Estrogen also has dual effects on PR expression, decreasing levels in the LE while at the same time increasing levels in the stroma and myometrium. Estrogen and P4 together mediate their effects in part through the ability of P4 to selectively inhibit myometrial ER expression while preserving GE expression. We also demonstrate a general negative feedback by P4 on PR expression that is most prominent in the GE. Finally, we demonstrate using the estrogen- and P4-responsive epithelial target gene LF that the differential regulation of PR in the glandular and luminal epithelium results in different functional responses of these compartments to P4. Together, our data indicate that the pleiotropic effects of estrogen and P4 in the adult mouse uterus are mediated by complex hormonal interregulation of ER and PR in specific uterine compartments.

The two-step isomerization of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) to Ins-1,3,4-P3 via the intermediate inositol 1,3,4,5-tetrakisphosphate (Ins-P4) was studied in intact RINm5F cells and in subcellular fractions. Muscarinic stimulation with carbamylcholine leads to a rapid (2 s) rise in both Ins-1,4,5-P3 and Ins-P4, whereas Ins-1,3,4-P3 was produced only after a lag of at least 5 s. In cells with depleted Ca2+ stores, the rise in Ins-1,4,5-P3 was nearly tripled, and that of Ins-1,3,4-P3 markedly diminished as compared to control cells. Raising the free Ca2+ concentration from 10(-7) to 10(-5) M increased inositol 1,4,5-triphosphate-3-kinase activity in cytosolic fractions by 2 1/2-fold (EC50 for Ca2+ approximately 0.8 microM) but had no effect on the activity of inositol 1,4,5-triphosphate-5-phosphomonoesterase. At 10(-7) M Ca2+ these two enzymes displayed comparable activity when assayed at concentrations of Ins-1,4,5-P3 occurring in stimulated cells; however, at 10(-5) M Ca2+, kinase activity predominates. These results suggest that Ins-1,4,5-P3 counter-regulates its own levels through the activity of inositol 1,4,5-trisphosphate 3-kinase and that the increase in [Ca2+]i may account for the transience of the rise in Ins-1,4,5-P3 seen during muscarinic stimulation of RINm5F cells.

The functional interaction ("cross-talk") of integrins with growth factor receptors has become increasingly clear as a basic mechanism in cell biology, defining cell growth, adhesion, and motility. However, no studies have addressed the microdomains in which such interaction takes place nor the effect of gangliosides and tetraspanins (TSPs) on such interaction. Growth of human embryonal WI38 fibroblasts is highly dependent on fibroblast growth factor (FGF) and its receptor (FGFR), stably associated with ganglioside GM3 and TSPs CD9 and CD81 in the ganglioside-enriched microdomain. Adhesion and motility of these cells are mediated by laminin-5 ((LN5) and fibronectin (FN) through alpha3beta1 and alpha5beta1 integrin receptors, respectively. When WI38 cells or its transformant VA13 cells were adhered to LN5 or FN, alpha3beta1 or alpha5beta1 were stimulated, giving rise to signaling to activate FGFR through tyrosine phosphorylation and inducing cell proliferation under serum-free conditions without FGF addition. Types and intensity of signaling during the time course differed significantly depending on the type of integrin stimulated (alpha3beta1 versus alpha5beta1), and on cell type (WI38 versus VA13). Such effect of cross-talk between integrins and FGFR was influenced strongly by the change of GM3 and TSPs. (i) GM3 depletion by P4 caused enhanced tyrosine phosphorylation of FGFR and Akt followed by MAPK activation, without significant change of ceramide level. GM3 depletion also caused enhanced co-immunoprecipitation of FGFR with alpha3/alpha5/beta1 and of these integrins with CD9/CD81. (ii) LN5- or FN-dependent proliferation of both WI38 and VA13 was strongly enhanced by GM3 depletion and by CD9/CD81 knockdown by siRNA. Thus, integrin-FGFR cross-talk is strongly influenced by GM3 and/or TSPs within the ganglioside-enriched microdomain.

Escherichia coli is an important bacterial species isolated from bovine mastitis. The rate of neutrophil recruitment into the mammary gland and their bactericidal activity largely affect the severity and outcome of the disease. Ketosis is a common metabolic disease, and affected dairy cows are known to have increased risk for mastitis and other infectious conditions. The disease is associated with high blood and milk levels of beta-hydroxybutyrate (BHBA), previously shown to negatively affect neutrophil function by unknown mechanisms. We show here that the mammary pathogenic E. coli strain P4 activates normal bovine neutrophils to form neutrophil extracellular traps (NETs), which are highly bactericidal against this organism. Preincubation of these neutrophils with increasing concentrations (0.1 to 8 mmol/liter) of BHBA caused a fivefold decrease of E. coli P4 phagocytosis, though intracellular killing was unaffected. Furthermore, BHBA caused a 10-fold decrease in the NETs formed by E. coli P4-activated neutrophils and a similar decrease in NET bactericidal activity against this organism. These negative effects of BHBA on bovine neutrophils might explain the increased susceptibility of ketotic cows to mastitis and other infectious conditions.

We used a single cell RNA-seq strategy to create an atlas of gene expression patterns in the developing kidney. At several stages of kidney development, histologically uniform populations of cells give rise to multiple distinct lineages. We performed single cell RNA-seq analysis of total mouse kidneys at E11.5 and E12.5, as well as the renal vesicles at P4. We define an early stage of progenitor cell induction driven primarily by gene repression. Surprising stochastic expression of marker genes associated with differentiated cell types was observed in E11.5 progenitors. We provide a global view of the polarized gene expression already present in the renal vesicle, the first epithelial precursor of the nephron. We show that Hox gene read-through transcripts can be spliced to produce intergenic homeobox swaps. We also identify a surprising number of genes with partially degraded noncoding RNA. Perhaps most interesting, at early developmental times single cells often expressed genes related to several developmental pathways. This provides powerful evidence that initial organogenesis involves a process of multilineage priming. This is followed by a combination of gene repression, which turns off the genes associated with most possible lineages, and the activation of increasing numbers of genes driving the chosen developmental direction.

Vesicle budding in eukaryotes depends on the activity of lipid translocases (P(4)-ATPases) that have been implicated in generating lipid asymmetry between the two leaflets of the membrane and in inducing membrane curvature. We show that Aminophospholipid ATPase3 (ALA3), a member of the P(4)-ATPase subfamily in Arabidopsis thaliana, localizes to the Golgi apparatus and that mutations of ALA3 result in impaired growth of roots and shoots. The growth defect is accompanied by failure of the root cap to release border cells involved in the secretion of molecules required for efficient root interaction with the environment, and ala3 mutants are devoid of the characteristic trans-Golgi proliferation of slime vesicles containing polysaccharides and enzymes for secretion. In yeast complementation experiments, ALA3 function requires interaction with members of a novel family of plant membrane-bound proteins, ALIS1 to ALIS5 (for ALA-Interacting Subunit), and in this host ALA3 and ALIS1 show strong affinity for each other. In planta, ALIS1, like ALA3, localizes to Golgi-like structures and is expressed in root peripheral columella cells. We propose that the ALIS1 protein is a beta-subunit of ALA3 and that this protein complex forms an important part of the Golgi machinery required for secretory processes during plant development.

Auditory vertex responses elicited by short tone bursts were compared with their magnetic counter parts. Special attention was paid to the behaviour of the N100 deflection of the response. Electrical responses were recorded from scalp locations Fp2, Fz, Cz, Pz, C4 and T4 and the magnetic responses half way between P4 and T6, at a point where the response has one of its amplitude extrema. Different ISIs (from 1 to 16 sec) were applied in order to differentiate specific and nonspecific evoked potential components from each other. The main results were as follows: (1) The scalp distsribution of the electical vertex response depends on the ISI used: with frequent stimulation there are no marked differences in the amplitudes of N100 between frontal and central areas but with long ISIs the amplitude maxima move to the vertex. (2) The magnetic responses also show a clear ISI dependence. The magnetic counterpart of N100 saturates at shorter ISIs than N100 recorded from the vertex. Independent of the ISI the magnetic counterpart of P200 is constantly very small. On the basis of the different sensitivities of the EEG and MEG to current sources of different orientations it is concluded that the auditory vertex response contains both modality specific and non-specific components. Experimental conditions, especially the ISI used, determine the relative contributions of these components to the potential recorded on the scalp.

There is evidence that antidepressant drug treatment during a critical period of postnatal development renders mice susceptible to depression- and anxiety-related behaviour in adulthood. The mechanism of how early antidepressant treatment brings about long-term effects in emotional behaviour is not yet understood, but neurotrophins, particularly brain-derived neurotrophic factor (BDNF), have been implicated in this context. We examined the long-term effects of a transient early postnatal fluoxetine treatment on depression- and anxiety-related behaviours as well as gene expression of BDNF and its receptor TrkB in C57BL/6J mice. Treatment with fluoxetine between postnatal days P4 and P21 resulted in a significant loss of body weight and long-lasting behavioural inhibition in adult mice in response to stressful events such as the light-dark or open field tests. Postnatal fluoxetine exposure also decreased behavioural despair in the forced swim test. Both body weight and behavioural alterations were restored by chronic fluoxetine treatment in adulthood. The behavioral alterations were accompanied by changes in hippocampal BDNF mRNA. Specifically, we show that early-life fluoxetine exposure resulted in the long-term upregulation of BDNF expression in adult mice. However, chromatin immunoprecipitation studies did not reveal any changes in the acetylation or trimethylation of histone H3 at the BDNF promoters. Our experiments show that behavioural and molecular changes induced by early postnatal fluoxetine administration are reversed by chronic fluoxetine treatment of adult mice to control levels.

It is well known that lipids are heterogeneously distributed throughout the cell. Most lipid species are synthesized in the endoplasmic reticulum (ER) and then distributed to different cellular locations in order to create the distinct membrane compositions observed in eukaryotes. However, the mechanisms by which specific lipid species are trafficked to and maintained in specific areas of the cell are poorly understood and constitute an active area of research. Of particular interest is the distribution of phosphatidylserine (PS), an anionic lipid that is enriched in the cytosolic leaflet of the plasma membrane. PS transport occurs by both vesicular and non-vesicular routes, with members of the oxysterol-binding protein family (Osh6 and Osh7) recently implicated in the latter route. In addition, the flippase activity of P4-ATPases helps build PS membrane asymmetry by preferentially translocating PS to the cytosolic leaflet. This asymmetric PS distribution can be used as a signaling device by the regulated activation of scramblases, which rapidly expose PS on the extracellular leaflet and play important roles in blood clotting and apoptosis. This review will discuss recent advances made in the study of phospholipid flippases, scramblases and PS-specific lipid transfer proteins, as well as how these proteins contribute to subcellular PS distribution.

BACKGROUND

Progesterone (P4) promotes its own secretion and the survival of human granulosa/luteal (GL) cells.

OBJECTIVE

The objective of these studies was to determine whether progesterone receptor membrane component-1 (PGRMC1) mediates P4's actions.

METHODS

In vitro studies were conducted on GL cells from women undergoing in vitro fertilization and GL5 cells, which are derived from GL cells.

METHODS

GL cells were obtained from women undergoing fertility treatment at a university-based clinic and used for in vitro studies.

METHODS

PCR, Western blot, and immunocytochemistry were used to detect various progestin binding proteins. (3)H-P4 binding kinetics were assessed on partially purified PGRMC1. Apoptosis was determined after culture by either TUNEL or DAPI staining. P4 was measured by an ELISA assay. PGRMC1 was depleted using small interfering RNA.

RESULTS

GL and GL5 cells expressed several P4 binding proteins including the nuclear progesterone receptor (PGR), progestin/adipoQ receptors (PAQR 7, 8, and 5) and PGRMC1. Ligand binding studies revealed that both P4 and the progestin, R5020, bound PGRMC1 with an EC(50) of approximately 10 nm. Interestingly, P4 inhibited apoptosis at concentrations in the 10 nm range, whereas R5020 stimulated P4 secretion at concentrations of at lease 16 mum. Depleting PGRMC1 attenuated P4's antiapoptotic action but failed to influence R5020-induced P4 secretion.

CONCLUSIONS

These studies conclusively demonstrate that in human GL cells PGRMC1 functions as a receptor through which P4 activates a signal cascade that prevents apoptosis. In contrast, PGRMC1 does not mediate P4's ability to acutely promote its own secretion.

The malaria parasite Plasmodium falciparum degrades hemoglobin in its acidic food vacuole for use as a major nutrient source. A novel metallopeptidase activity, falcilysin, was purified from food vacuoles and characterized. Falcilysin appears to function downstream of the aspartic proteases plasmepsins I and II and the cysteine protease falcipain in the hemoglobin proteolytic pathway. It is unable to cleave hemoglobin or denatured globin but readily destroys peptide fragments of hemoglobin. Falcilysin cleavage sites along the alpha and beta chains of hemoglobin are polar in character, with charged residues located in the P1 and/or P4' positions. In contrast, plasmepsins I and II and falcipain prefer hydrophobic residues around the scissile bond. The gene encoding falcilysin has been cloned. Its coding sequence exhibits features characteristic of clan ME family M16 metallopeptidases, including an "inverted" HXXEH active site motif. Falcilysin shares primary structural features with M16 family members such as insulysin, mitochondrial processing peptidase, nardilysin, and pitrilysin as well as with data base hypothetical proteins that are potential M16 family members. The characterization of falcilysin increases our understanding of hemoglobin catabolism in P. falciparum and the unusual M16 family of metallopeptidases.

B lymphopoiesis begins in the fetal liver, switching after birth to the bone marrow, where it persists for life. The unique developmental outcomes of each phase are well documented, yet their molecular requirements are not. Here we describe two allelic X-linked mutations in mice that caused cell-intrinsic arrest of adult B lymphopoiesis. Mutant fetal liver progenitors generated B cells in situ but not in irradiated adult bone marrow, which emphasizes a necessity for the affected pathway only in the context of adult bone marrow. The causative mutations were ascribed to Atp11c, which encodes a P4-type ATPase with no previously described function. Our data establish an essential, cell-autonomous and context-sensitive function for ATP11C, a putative aminophospholipid flippase, in B cell development.

Endocytic sorting is achieved through the formation of morphologically and functionally distinct sub-domains within early endosomes. Cargoes destined for recycling are sorted to and transported through newly-formed tubular membranes, but the processes that regulate membrane tubulation are poorly understood. Here, we identified a novel Caenorhabditis elegans Cdc50 family protein, CHAT-1, which acts as the chaperone of the TAT-1 P4-ATPase to regulate membrane phosphatidylserine (PS) asymmetry and endocytic transport. In chat-1 and tat-1 mutants, the endocytic sorting process is disrupted, leading to defects in both cargo recycling and degradation. TAT-1 and CHAT-1 colocalize to the tubular domain of the early endosome, the tubular endocytic recycling compartment (ERC), and the recycling endosome where PS is enriched on the cytosolic surface. Loss of tat-1 and chat-1 function disrupts membrane PS asymmetry and abrogates the tubular membrane structure. Our data suggest that CHAT-1 and TAT-1 maintain membrane phosphatidylserine asymmetry, thus promoting membrane tubulation and regulating endocytic sorting and recycling.