This experiment provides brain event-related potential (ERP) evidence for differential processing of visually presented pleasant and unpleasant affectively valent words (mood adjectives) for both supraliminal (40 ms) and subliminal (unmasked, 1 ms) stimulus durations. Unpleasant words elicited a more positive amplitude than pleasant words in both durations. ERP components (P1, N1, P2, P3, and a late positive potential; LP) were measured at six electrode sites (F3, F4, P3, P4, CzPz, Oz). ERPs to subliminal stimuli demonstrated differences between pleasant and unpleasant words in the left hemisphere across all measured components. Supraliminal processing showed similar differences in the left hemisphere for early components (P1 and N1), but bilateral differences for late components (P3 and LP). Activity in the P2 time window was associated with the divergence between supraliminal and subliminal affective responses. Implications for the study of affect and consciousness are discussed.

Progesterone (P4) signaling through its nuclear transcription factor, the progesterone receptor (PR), is essential for normal uterine function. Although deregulation of PR-mediated signaling is known to underscore uterine dysfunction and a number of endometrial pathologies, the early molecular mechanisms of this deregulation are unclear. To address this issue, we have defined the genome-wide PR cistrome in the murine uterus using chromatin immunoprecipitation (ChIP) followed by massively parallel sequencing (ChIP-seq). In uteri of ovariectomized mice, we identified 6367 PR-binding sites in the absence of P4 ligand; however, this number increased at nearly 3-fold (18,432) after acute P4 exposure. Sequence analysis revealed that approximately 73% of these binding sites contain a progesterone response element or a half-site motif recognized by the PR. Many previously identified P4 target genes known to regulate uterine function were found to contain PR-binding sites, confirming the validity of our methodology. Interestingly, when the ChIP-seq data were coupled with our microarray expression data, we identified a novel regulatory role for uterine P4 in circadian rhythm gene expression, thereby uncovering a hitherto unexpected new circadian biology for P4 in this tissue. Further mining of the ChIP-seq data revealed Sox17 as a direct transcriptional PR target gene in the uterus. As a member of the Sox transcription factor family, Sox17 represents a potentially novel mediator of PR action in the murine uterus. Collectively, our first line of analysis of the uterine PR cistrome provides the first insights into the early molecular mechanisms that underpin normal uterine responsiveness to acute P4 exposure. Future analysis promises to reveal the PR interactome and, in turn, potential therapeutic targets for the diagnosis and/or treatment of endometrial dysfunction.

The antitumor drug miltefosine has been recently approved as the first oral drug active against visceral leishmaniasis. We have previously identified the L. donovani miltefosine transporter (LdMT) as a P-type ATPase involved in phospholipid translocation at the plasma membrane of Leishmania parasites. Here we show that this protein is essential but not sufficient for the phospholipid translocation activity and, thus, for the potency of the drug. Based on recent findings in yeast, we have identified the putative beta subunit of LdMT, named LdRos3, as another protein factor required for the translocation activity. LdRos3 belongs to the CDC50/Lem3 family, proposed as likely beta subunits for P4-ATPases. The phenotype of LdRos3-defective parasites was identical to that of the LdMT-/-, including a defect in the uptake of 7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)-phosphatidylserine, generally considered as not affected in Lem3p-deficient yeast. Both LdMT and LdRos3 normally localized to the plasma membrane but were retained inside the endoplasmic reticulum in the absence of the other protein or when inactivating point mutations were introduced in LdMT. Modulating the expression levels of either protein independently, we show that any one of them could behave as the protein limiting the level of flippase activity. Thus, LdMT and LdRos3 seem to form part of the same translocation machinery that determines flippase activity and miltefosine sensitivity in Leishmania, further supporting the consideration of CDC50/Lem3 proteins as beta subunits required for the normal functioning of P4-ATPases.

The histone-like dimeric HU protein of Escherichia coli is encoded by two closely related genes, hupA and hupB. We show here that expression from the single hupA promoter and from the three hupB promoters varies during growth phase. The weak hupB-P4 promoter is active immediately after dilution. Transcription of the hupA gene is activated early in logarithmic phase. A little later, at mid to late exponential phase, RNA originating at the hupB-P2 promoter is detected. The hupB-P3 promoter is activated last when the cells enter stationary phase. Although the hup mRNAs are unstable, the HU protein is very stable so that the variations in the mRNAs synthesis are reflected in the level of the two HU subunits and in the composition of HU dimers. Cells growing exponentially contain a mixture of homodimeric alpha 2 and heterodimeric alpha beta but no beta 2 is detected. In stationary cells, the predominant form is the heterodimer alpha beta. The presence of the heterodimeric form is required for optimal survival of E. coli after prolonged starvation. The three forms of HU are not equivalent, since beta 2 is incapable of promoting formation of DNA supercoiling like alpha beta and alpha 2 do. The putative roles of each form of HU are discussed.

OBJECTIVE

To characterize equine adipose tissue-derived stromal cell (ASC) frequency and growth characteristics and assess of their adipogenic and osteogenic differentiation potential.

METHODS

In vitro experimental study.

METHODS

Horses (n=5; aged, 9 months to 5 years).

METHODS

Cell doubling characteristics of ASCs harvested from supragluteal subcutaneous adipose tissue were evaluated over 10 passages. Primary, second (P2), and fourth (P4) passage ASCs were induced under appropriate conditions to undergo adipogenesis and osteogenesis. Limit dilution assays were performed on each passage to determine the frequency of colony-forming units with a fibroblastic (CFU-F) phenotype and the frequency of ASC differentiation into the adipocyte (CFU-Ad) and osteoblast (CFU-Ob) phenotype.

RESULTS

ASC isolates exhibited an average cell-doubling time of 2.1+/-0.9 days during the first 10 cell doublings. Approximately 1 in 2.3+/-0.4 of the total stromal vascular fraction nucleated cells were ASCs, based on the CFU-F assays, and 1 in 3.6+/-1.3 expressed alkaline phosphatase, an osteogenic marker. Primary ASCs differentiated in response to adipogenic (1 in 4.9+/-5.4, CFU-Ad) and osteogenic (1 in <2.44, CFU-Ob) inductive conditions and maintained their differentiation potential during subsequent passages (P2 and P4).

CONCLUSIONS

The frequency, in vitro growth rate, and adipogenic and osteogenic differentiation potential of equine ASCs show some differences to those documented for ASCs in other mammalian species.

CONCLUSIONS

Adipose tissue is a potential source of adult stem cells for tissue engineering applications in equine veterinary medicine.

Seven isoflavones, biochanin A, daidzein, genistein, genistin, prunectin, puerarin, and pseudobaptigenin were tested for cytostatic and cytotoxic effects on 10 newly established cancer cell lines of the human gastrointestinal origin. Proliferation of HSC-41E6, HSC-45M2, and SH101-P4 stomach cancer cell lines was strongly inhibited by biochanin A and genistein, whereas other stomach, esophageal, and colon cancer lines were moderately suppressed by both compounds. Biochanin A and genistein were cytostatic at low concentrations (< 20 micrograms/ml for biochanin A, < 10 micrograms/ml for genistein) and were cytotoxic at higher concentrations >> 40 micrograms/ml for biochanin A,>> 20 micrograms/ml for genistein). DNA fragmentation was observed at cytotoxic doses of both compounds, indicating the apoptotic mode of cell death by the compounds. Chromatin condensation and nuclear fragmentation of each cell line were also observed. The advent of apoptosis was dose dependent for both isoflavones. Biochanin A suppressed tumor growth of HSC-45M2 and HSC-41E6 lines in athymic nude mice. Our results suggest that two of isoflavone derivatives, biochanin A and genistein, inhibit the cell growth of stomach cancer cell lines in vitro through activation of a signal transduction pathway for apoptosis. Moreover, in vivo experiments demonstrate that biochanin A can be used as an anticancer agent.

When linear double-stranded DNA is packed inside bacteriophage capsids, it becomes highly compacted. However, the phage is believed to be fully effective only if the DNA is not entangled. Nevertheless, when DNA is extracted from a tailless mutant of the P4 phage, DNA is found to be cyclic and knotted (probability of 0.95). The knot spectrum is very complex, and most of the knots have a large number of crossings. We quantified the frequency and crossing numbers of these knots and concluded that, for the P4 tailless mutant, at least half the knotted molecules are formed while the DNA is still inside the viral capsid rather than during extraction. To analyze the origin of the knots formed inside the capsid, we compared our experimental results to Monte Carlo simulations of random knotting of equilateral polygons in confined volumes. These simulations showed that confinement of closed chains to tightly restricted volumes results in high knotting probabilities and the formation of knots with large crossing numbers. We conclude that the formation of the knots inside the viral capsid is driven mainly by the effects of confinement.

BACKGROUND

JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a approximately 1 kb CLN3 mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated Cln3Deltaex7/8 knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology.

RESULTS

To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized CbCln3 wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous CbCln3Deltaex7/8 precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced.

CONCLUSIONS

These findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival.

The interferon-induced promyelocytic leukaemia (PML) protein localizes both in the nucleoplasm and in matrix-associated multi-protein complexes known as nuclear bodies (NBs). NBs are disorganized in acute promyelocytic leukaemia or during some viral infections, suggesting that PML NBs could be a part of cellular defense mechanism. Rabies virus, a member of the rhabdoviridae family, replicates in the cytoplasm. Rabies phosphoprotein P and four other amino-terminally truncated products (P2, P3, P4, P5) are all translated from P mRNA. P and P2 are located in the cytoplasm, whereas P3, P4 and P5 are found mostly in the nucleus. Infection with rabies virus reorganized PML NBs. PML NBs became larger and appeared as dense aggregates when analysed by confocal or electron microscopy, respectively. The expression of P sequesters PML in the cytoplasm where both proteins colocalize, whereas that of P3 results in an increase in PML body size, as observed in infected cells. The P and P3 interacted directly in vivo and in vitro with PML. The C-terminal domain of P and the PML RING finger seem to be involved in this binding. Moreover, PML-/- primary mouse embryonic fibroblasts expressed viral proteins at a higher level and produced 20 times more virus than wild-type cells, suggesting that the absence of all PML isoforms resulted in an increase in rabies virus replication.

Mouse retinal photoreceptor cell generation and morphogenesis take place in a well-characterized temporal sequence. Both rod and cone photoreceptor differentiation and synaptogenesis occur postnatally, but the relative timing of these events has been difficult to document due to the paucity of cell-specific markers. We have found that antibodies to neuron-specific enolase (NSE) preferentially label a subpopulation of photoreceptors in the outer nuclear layer (ONL) of the mouse retina in addition to labeling ganglion, amacrine, bipolar, and horizontal cells within the inner layers of the retina. The appearance of NSE immunoreactivity in the different classes of retinal neurons during development showed a close temporal relationship to the onset of expression of the synaptic vesicle-associated protein SV2 and clearly preceded the sequential development of synaptic connections in both inner and outer synaptic layers. The NSE-immunoreactive photoreceptors were identified as cones by dual labeling of their inner segments with the lectin peanut agglutinin or by colabeling with antisera to cone photopigments. Axonal extensions of NSE-labeled cone cells were shown to interact with those of differentiating horizontal cells as early as postnatal day 3 (P3). Colocalization of NSE with SV2 indicated that cone cells began to make synaptic contacts with horizontal cell processes several days prior to the development of rod synaptic terminals. Between P4 and P11, cone photoreceptor cell nuclei were observed to be scattered at various levels throughout the ONL and thus appeared to have become displaced from their previous position directly beneath the outer limiting membrane (OLM). By P12, the cone nuclei had migrated sclerad once again and were now observed to be neatly aligned adjacent to the OLM. In the rd mouse mutant, this migratory process was delayed, so that, at P12, positioning of the cone cell nuclei within the ONL was still quite irregular. Thus, we have identified a late migratory phase for cone photoreceptors during the second week after birth that correlates with the timing of maturation of the rod synaptic terminals just prior to eye opening. The types of cues used by maturing cone cells for their eventual sclerad location remain to be elucidated.

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus. Viral maturation requires a main protease (3CL(pro)) to cleave the virus-encoded polyproteins. We report here that the 3CL(pro) containing additional N- and/or C-terminal segments of the polyprotein sequences undergoes autoprocessing and yields the mature protease in vitro. The dimeric three-dimensional structure of the C145A mutant protease shows that the active site of one protomer binds with the C-terminal six amino acids of the protomer from another asymmetric unit, mimicking the product-bound form and suggesting a possible mechanism for maturation. The P1 pocket of the active site binds the Gln side chain specifically, and the P2 and P4 sites are clustered together to accommodate large hydrophobic side chains. The tagged C145A mutant protein served as a substrate for the wild-type protease, and the N terminus was first digested (55-fold faster) at the Gln(-1)-Ser1 site followed by the C-terminal cleavage at the Gln306-Gly307 site. Analytical ultracentrifuge of the quaternary structures of the tagged and mature proteases reveals the remarkably tighter dimer formation for the mature enzyme (K(d) = 0.35 nm) than for the mutant (C145A) containing 10 extra N-terminal (K(d) = 17.2 nM) or C-terminal amino acids (K(d) = 5.6 nM). The data indicate that immature 3CL(pro) can form dimer enabling it to undergo autoprocessing to yield the mature enzyme, which further serves as a seed for facilitated maturation. Taken together, this study provides insights into the maturation process of the SARS 3CL(pro) from the polyprotein and design of new structure-based inhibitors.

Using a combination of gel filtration and anion exchange FPLC, three different RNA polymerase holoenzymes from Streptomyces coelicolor A3(2) have been separated. Each holoenzyme transcribes from only one of the four promoters of the S. coelicolor A3(2) dagA gene. Holoenzyme reconstitution experiments identified the sigma factors responsible for recognition of two of the promoters. The previously identified E sigma 49 transcribes from the dagA p3 promoter, whereas a novel species, E sigma 28, recognizes the dagA p2 promoter. Circumstantial evidence suggests that the third holoenzyme, which transcribes from the dagA p4 promoter, is the previously identified E sigma 35. This level of transcriptional complexity supports the idea that RNA polymerase heterogeneity may play a central role in the regulation and coordination of gene expression in this biochemically and morphologically complex bacterium.

The thalamocortical projection to rodent somatosensory ("barrell") cortex is highly ordered in both the radial and the tangential dimensions. During a brief period of postnatal development, thalamocortical axons establish two tiers of terminations, in the deep layers and in layer IV, and form whisker-specific clusters within layer IV; however, little is known about the cues that guide them to their appropriate radial and tangential positions. To gain insight into potential mechanisms underlying this process, we studied the development of thalamocortical termination patterns in mouse barrel cortex at high spatial resolution. Developing thalamocortical axons were labeled in fixed slices with the lipophilic carbocyanine dye Dil and imaged with a laser scanning confocal microscope. On the day of birth (postnatal day 0, P0) axons coursed through layers VI and V, with little or no branching. By P2 the lower tier of terminations, at the border of layers VI and V, was clearly identifiable. Below this tier axons coursed obliquely or tangentially, forming a dense meshwork of intersecting fibers, but with no apparent branching. By P4 the upper tier of terminations, in layer IV, was clearly recognizable, and consisted of periodic, dense clusters of terminal arborizations. In marked contrast to the oblique and apparently disorderly course followed by axons in layer VI and lower layer V, axons in upper layer V heading toward the upper tier were organized in loose bundles running radially, suggesting that axons destined to terminate in a particular layer IV barrel had already reached their appropriate tangential coordinates within the lower tier. Thus, the pattern of thalamocortical terminations in layer IV seems to be projected from the deep tier of terminations, and does not develop from an initially profuse arborization pattern through pruning of inappropriate branches.

We report here the large scale purification of DNA topoisomerase II from calf thymus glands, using the unknotting of naturally knotted P4 phage DNA as an assay for enzymatic activity. Topoisomerase II was purified more than 1300-fold as compared to the whole cell homogenate, with 22% yield. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 125 and 140 kDa. Tryptic maps of the two bands indicated that they derive from the same protein. Using these fragments, specific polyclonal antisera to topoisomerase II were raised in rabbits. Immunoblotting of whole cell lysates from various species indicated that topoisomerase II is well conserved among mammals and has a native subunit molecular mass of 180 kDa. Analytical sedimentation and gel filtration were used to determine a sedimentation coefficient of 9.8 S and a Stokes radius of 68 A. The calculated solution molecular mass of 277 kDa implies a dimer structure in solution. The purified topoisomerase II unknots P4 DNA in an ATP-dependent manner and is highly stimulated in its relaxation activity by ATP. A DNA-stimulated ATPase activity, as has been found with other type II topoisomerases, is associated with the purified enzyme. Approximate kinetic parameters for the ATPase reaction were determined to be: a Vmax of 0.06 nmol of ATP/(micrograms of protein) (min) and Km of 0.2 mM in the absence of DNA, and a Vmax of 0.2 nmol of ATP/(micrograms of protein) (min) and Km of 0.4 mM ATP in the presence of supercoiled plasmid DNA.

Early in development, the depolarizing GABA(A)ergic signaling is needed for normal neuronal differentiation. It is shown here that hyperpolarizing reversal potentials of GABA(A)ergic postsynaptic currents (E(GABA)) appear earlier in female than in male rat CA1 pyramidal neurons because of increased potassium chloride cotransporter 2 (KCC2) expression and decreased bumetanide-sensitive chloride transport in females. Three episodes of neonatal kainic acid-induced status epilepticus (3KA-SE), each elicited at postnatal days 4 (P4)-P6, reverse the direction of GABA(A)ergic responses in both sexes. In males, 3KA-SE trigger a premature appearance of hyperpolarizing GABA(A)ergic signaling at P9, instead of P14. This is driven by an increase in KCC2 expression and decrease in bumetanide-sensitive chloride cotransport. In 3KA-SE females, E(GABA) transiently becomes depolarizing at P8-P13 because of increase in the activity of a bumetanide-sensitive NKCC1 (sodium potassium chloride cotransporter 1)-like chloride cotransporter. However, females regain their hyperpolarizing GABA(A)ergic signaling at P14 and do not manifest spontaneous seizures in adulthood. In maternally separated stressed controls, a hyperpolarizing shift in E(GABA) was observed in both sexes, associated with decreased bumetanide-sensitive chloride cotransport, whereas KCC2 immunoreactivity was increased in males only. GABA(A) receptor blockade at the time of 3KA-SE or maternal separation reversed their effects on E(GABA). These data suggest that the direction of GABA(A)-receptor signaling may be a determining factor for the age and sex-specific effects of prolonged seizures in the hippocampus, because they relate to normal brain development and possibly epileptogenesis. These effects differ from the consequences of severe stress.

The female hormone progesterone (P4) promotes the expansion of stem-like cancer cells in estrogen receptor (ER)- and progesterone receptor (PR)-positive breast tumors. The expanded tumor cells lose expression of ER and PR, express the tumor-initiating marker CD44, the progenitor marker cytokeratin 5 (CK5) and are more resistant to standard endocrine and chemotherapies. The mechanisms underlying this hormone-stimulated reprogramming have remained largely unknown. In the present study, we investigated the role of microRNAs in progestin-mediated expansion of this dedifferentiated tumor cell population. We demonstrate that P4 rapidly downregulates miR-29 family members, particularly in the CD44(+) cell population. Downregulation of miR-29 members potentiates the expansion of CK5(+) and CD44(+) cells in response to progestins, and results in increased stem-like properties in vitro and in vivo. We demonstrate that miR-29 directly targets Krüppel-like factor 4 (KLF4), a transcription factor required for the reprogramming of differentiated cells to pluripotent stem cells, and for the maintenance of breast cancer stem cells. These results reveal a novel mechanism, whereby progestins increase the stem cell-like population in hormone-responsive breast cancers, by decreasing miR-29 to augment PR-mediated upregulation of KLF4. Elucidating the mechanisms whereby hormones mediate the expansion of stem-like cells furthers our understanding of the progression of hormone-responsive breast cancers.

HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens.

Progesterone's (P4) negative feedback actions in the female reproductive axis are exerted in part by suppression of hypothalamic GnRH release. Here we show that P4 can inhibit GnRH release by a mechanism independent of a nuclear P4 receptor (PR(A/B)). Injections of P4, but not vehicle, allopregnanolone, or dexamethasone, acutely suppressed LH levels in both wild-type and P4 receptor knockout ovariectomized mice; pituitary responsiveness to GnRH was retained during P4 treatment, indicating a hypothalamic action. Superfusion of GnRH-producing GT1-7 cells with medium containing 10(-7) m P4 produced a rapid reduction in GnRH release. Incubation with P4 (10(-9) to 10(-7) M) inhibited forskolin-stimulated cAMP accumulation; cotreatment with pertussis toxin prevented this effect. Treatment of GT1-7 cell membranes with P4 caused activation of an inhibitory G protein (G(i)), as shown by immunoprecipitation with a G(i) antibody of most of the increase in membrane-bound [(35)S]GTPgamma-S. Saturation binding analyses demonstrated the presence of a high affinity (K(d) 5.85 nM), limited capacity (Bmax 62.2 nM) binding site for P4. RT-PCR analysis revealed the presence of mRNAs encoding both isoforms of the membrane P4 receptors, mPRalpha and mPRbeta. Western blotting, immunocytochemistry, and flow cytometry experiments similarly revealed expression of mPR proteins in the plasma membranes of GT1-7 cells. Treatment with mPRalpha siRNA attenuated specific P4 binding to GT1-7 cell membranes and reversed the P4 inhibition of cAMP accumulation. Taken together, our results suggest that negative feedback actions of P4 include rapid PR(A/B)-independent effects on GnRH release that may in part be mediated by mPRs.

Adipose tissue is a structure highly specialized in energy storage. The adipocyte is the parenchymal component of adipose tissue and is known to be mesoderm or neuroectoderm in origin; however, adipocyte development remains poorly understood. Here, we investigated the development of adipose tissue by analyzing postnatal epididymal adipose tissue (EAT) in mouse. EAT was found to be generated from non-adipose structure during the first 14 postnatal days. From postnatal day 1 (P1) to P4, EAT is composed of multipotent progenitor cells that lack adipogenic differentiation capacity in vitro, and can be regarded as being in the 'undetermined' state. However, the progenitor cells isolated from P4 EAT obtain their adipogenic differentiation capacity by physical interaction generated by cell-to-matrix and cell-to-cell contact both in vitro and in vivo. In addition, we show that impaired angiogenesis caused by either VEGFA blockade or macrophage depletion in postnatal mice interferes with adipose tissue development. We conclude that appropriate interaction between the cellular and matrix components along with proper angiogenesis are mandatory for the development of adipose tissue.

In contrast to our knowledge about the anatomical development of the mammalian central auditory system, the development of its physiological properties is still poorly understood. In order to better understand the physiological properties of the developing mammalian auditory brainstem, we made intracellular recordings in brainstem slices from perinatal rats to examine synaptic transmission in the superior olivary complex, the first binaural station in the ascending auditory pathway. We concentrated on neurons in the lateral superior olive (LSO), which in adults, are excited from the ipsilateral side and inhibited from the contralateral side. Already at embryonic day (E) 18, when axon collaterals begin to invade the LSO anlage, synaptic potentials could be evoked from ipsilateral, as well as from contralateral inputs. Ipsilaterally elicited PSPs were always depolarizing, regardless of age. They had a positive reversal potential and could be completely blocked by the non-NMDA glutamate receptor antagonist CNQX. In contrast, contralaterally elicited PSPs were depolarizing from E18-P4, yet they turned into "adult-like," hyperpolarizing PSPs after P8. Their reversal potential shifted dramatically from -21.6 +/- 17.7 mV (E18-P0) to -73.0 +/- 7.1 mV (P10). Regardless of their polarity, contralaterally elicited PSPs were reversibly blocked by the glycine receptor antagonist strychnine. Bath application of glycine and its agonist beta-alanine further confirmed the transitory depolarizing action of glycine in the auditory brainstem. Since the transient excitatory behavior of glycine occurs during a period during which glycinergic synaptic connections in the LSO are refined by activity-dependent mechanisms, glycinergic excitation might be a mechanism by which synaptic rearrangement in the contralateral inhibitory pathway is accomplished.

Digitonin, a sterol glycoside which complexes with cholesterol, stripped off the envelope of vesicular stomatitis (VS) virions and liberated two viral structural proteins, 83% of P6 and 53% of P4. Deoxycholate also disrupted VS virions but released nucleocapsid cores which could be identified by higher buoyant density, ratio of incorporated (3)H-uridine to (14)C-protein, and electron microscopy. The major nucleocapsid protein was P5 but varying amounts of the minor protein aggregate P2 were present, depending on the concentration of urea used for extraction. P2 appeared to be a polymer of P5. Two other minor structural proteins, P1 and P3, could not be located in the virion. From these data, we conclude that the three microscopically identifiable structures of VS virions are each composed primarily of a single major protein, as follows: P6 = envelope protein, P4 = protein of underlying "shell," and P5 = nucleocapsid protein.

Overproduction of the exopolysaccharide alginate causes mucoid colony morphology in Pseudomonas aeruginosa and is considered a major virulence determinant expressed by this organism during chronic respiratory infections in cystic fibrosis. One of the principal regulatory elements governing conversion to mucoidy in P. aeruginosa is AlgU, an alternative sigma factor which is 66% identical to and functionally interchangeable with sigma E from Escherichia coli and Salmonella typhimurium. sigma E has been implicated in the expression of systems enhancing bacterial resistance to environmental stress. In this study, we report that the gene encoding AlgU is transcribed in wild-type nonmucoid P. aeruginosa from multiple promoters (P1 through P5) that fall into three categories: (i) the P1 and P3 promoters, which display strong similarity to the -35 and -10 canonical sequences of sigma E promoters and were found to be absolutely dependent on AlgU; (ii) the P2 promoter, which was less active in algU mutants, but transcription of which was not completely abrogated in algU::Tcr cells; and (iii) the transcripts corresponding to P4 and P5, which were not affected by inactivation of algU. Introduction of E. coli rpoE (encoding sigma E) or algU into P. aeruginosa algU::Tcr strains restored P1 and P3 transcription and brought the P2 signal back to the wild-type level. The AlgU-dependent promoters P1 and P3 were inducible by heat shock in wild-type nonmucoid P. aeruginosa PAO1. At the protein level, induction of AlgU synthesis under conditions of extreme heat shock was detected by metabolic labeling of newly synthesized proteins, two-dimensional gel analysis, and reaction with polyclonal antibodies raised against an AlgU peptide. Another AlgU-dependent promoter, the proximal promoter of algR, was also found to be induced by heat shock. Under conditions of high osmolarity, growth at elevated temperature induced alginate synthesis in the wild-type nonmucoid P. aeruginosa PAO1. Cumulatively, these results suggest that algU itself is subject to complex regulation and is inducible by extreme heat shock, that the alginate system is a subset of the stress-responsive elements controlled by AlgU, and that AlgU and, by extension, its homologs in other organisms (e.g., sigma E in S. typhimurium) may play a role in bacterial virulence and adjustments to adverse growth conditions.

Research in recent years on plant Major Intrinsic Proteins (MIPs), commonly referred to as 'aquaporins', has seen a vast expansion in the substrates found to be transported via these membrane channels. The diversity in sizes, chemical nature and physiological significance of these substrates has meant a need to critically analyse the possible structural and biochemical properties of MIPs that transport these, in order to understand their roles. In this work we have undertaken a comprehensive analysis of all plant MIPs, coming from different families, that have been proven to transport ammonia, boron, carbon dioxide, hydrogen peroxide, silicon and urea. The sequences were analysed for all primary selectivity-related motifs (NPA motifs, ar/R filter, P1-P5 residues). In addition, the putative regulatory phosphorylation and glycosylation sites and mechanistic regulators such as loop lengths have been analysed. Further, nine specificity-determining positions (SDPs) were predicted for each group. The results show the ar/R filter residues, P2-P4 positions and some of the SDPs are characteristic for certain groups, and O-glycosylation sites are unique to a subgroup while N-glycosylation was characteristic of the other MIPs. Certain residues, especially in loop C, and structural parameters such as loop lengths also contribute to the uniqueness of groups. The comprehensive analysis makes significant inroads into appraising the intriguing diversity of plant MIPs and their roles in fundamental life processes, and provides tools for plant selections, protein engineering and transgenics.

We propose an innovative, integrated, cost-effective health system to combat major non-communicable diseases (NCDs), including cardiovascular, chronic respiratory, metabolic, rheumatologic and neurologic disorders and cancers, which together are the predominant health problem of the 21st century. This proposed holistic strategy involves comprehensive patient-centered integrated care and multi-scale, multi-modal and multi-level systems approaches to tackle NCDs as a common group of diseases. Rather than studying each disease individually, it will take into account their intertwined gene-environment, socio-economic interactions and co-morbidities that lead to individual-specific complex phenotypes. It will implement a road map for predictive, preventive, personalized and participatory (P4) medicine based on a robust and extensive knowledge management infrastructure that contains individual patient information. It will be supported by strategic partnerships involving all stakeholders, including general practitioners associated with patient-centered care. This systems medicine strategy, which will take a holistic approach to disease, is designed to allow the results to be used globally, taking into account the needs and specificities of local economies and health systems.