During retinal development, retinal vascularization begins in the inner retinal layer and sprouts radially from the optic nerve to reach the periphery of the retina. Subsequently, retinal vessels sprout into the deep retinal layer to form three parallel of the nerve fiber layer and two plexiform layers. In this process, endothelial cells are closely related to astrocytes and pericytes with strict chronological order. Here, we provide that the recruitment of pericytes and astrocytes to vascular tube of endothelial cells is closely associated with the formation of tight junction in developing retinal vessels. At P4, endothelial cells of retinal vessels behind the invading front directly contact to pericytes, but not to foot processes of astrocytes, where ZO-1 was already weakly immunoreactive along retinal endothelial cells. With the progression of retinal development, foot processes of astrocytes are gathered around retinal vessels and the maturation of tight junction in endothelial cells is clearly defined, which was temporally and spatially accordant to the expression of a tight junction protein, ZO-1. In addition, tight junction could be formed with contact of pericytes to endothelial cells without the prominent ensheathment of astrocytic foot processes, which coincided with the appearance of a tight junction protein, ZO-1. Therefore, these data demonstrate that the tight junction of endothelial cells in the blood-retina barrier could be developed by cellular interactions between pericytes, astrocytes, and endothelial cells. Moreover, ZO-1 as well as occludin or claudin might demonstrate the tightness of blood-retina barrier in developing retina.

BACKGROUND

Retinal bipolar cells comprise a diverse group of neurons. Cone bipolar cells and rod bipolar cells are so named for their connections with cone and rod photoreceptors, respectively. Morphological criteria have been established that distinguish nine types of cone bipolar cells and one type of rod bipolar cell in mouse and rat. While anatomical and physiological aspects of bipolar types have been actively studied, little is known about the sequence of events that leads to bipolar cell type specification and the potential relationship this process may have with synapse formation in the outer plexiform layer. In this study, we have examined the birth order of rod and cone bipolar cells in the developing mouse and rat in vivo.

RESULTS

Using retroviral lineage analysis with the histochemical marker alkaline phosphatase, the percentage of cone and rod bipolar cells born on postnatal day 0 (P0), P4, and P6 were determined, based upon the well characterized morphology of these cells in the adult rat retina. In this in vivo experiment, we have demonstrated that cone bipolar genesis clearly precedes rod bipolar genesis. In addition, in the postnatal mouse retina, using a combination of tritiated-thymidine birthdating and immunohistochemistry to distinguish bipolar types, we have similarly found that cone bipolar genesis precedes rod bipolar genesis. The tritiated-thymidine birthdating studies also included quantification of the birth of all postnatally generated retinal cell types in the mouse.

CONCLUSIONS

Using two independent in vivo methodologies in rat and mouse retina, we have demonstrated that there are distinct waves of genesis of the two major bipolar cell types, with cone bipolar genesis preceding rod bipolar genesis. These waves of bipolar genesis correspond to the order of genesis of the presynaptic photoreceptor cell types.

4-Vinylcyclohexene diepoxide (VCD) causes early, gradual ovarian failure in mice because it specifically targets small pre-antral ovarian follicles. The period between loss of these follicles and ovarian failure is analogous to perimenopause in women. We sought to characterize the period of onset of ovarian failure in VCD-treated mice in regard to estrous cycle length and hormonal changes. Female C57Bl/6 mice (age, 28 days) were dosed daily for 15 days with VCD (160 mg/kg intraperitoneally) to cause early ovarian failure or with vehicle only (control animals). Cycle length was monitored by vaginal cytology. Plasma levels of 17beta-estradiol (E2), progesterone (P4), and follicle-stimulating hormone (FSH) in control and VCD-treated animals were measured during proestrus of cycles 1 through 12. Cycle length (mean, 5.8 days) did not differ between groups for cycles 1 through 4. In contrast, cycle length during cycles 5 through 12 was increased (mean length, 10.9 days; P < 0.05 versus control) in VCD-treated animals, which also showed an apparent increase in plasma FSH levels. Plasma E2 and P4 at proestrus did not differ between groups during any cycle. Ovarian failure in VCD-treated mice was confirmed by histological evaluation on day 156 after onset of dosing, whereas control animals were still cycling. Therefore, despite compromised cycle length in VCD-treated mice, peak ovarian steroid production in preovulatory follicles at proestrus is adequate. These results demonstrate that the VCD-treated mouse can serve as an appropriate model to mimic hormonal changes during the perimenopausal transition in women.

To investigate the developmental fates and the migratory pathways of dividing progenitors in both the white matter (WM) and the external granule layer (EGL) in the early postnatal rat cerebellum, a replication-deficient retrovirus carrying the beta-galactosidase gene (BAG) was injected into the deep cerebellar tissue or the EGL of postnatal rats to label dividing progenitors. After 1-3 days post-injection (1-3 dpi) of BAG into the deep cerebellar tissue of postnatal day 4/5 (P4/5) rats, labeled immature, unipolar cells were found mainly in the WM. From 4 to 6 dpi, similar cells appeared in the internal granule (IGL), Purkinje cell, and molecular layers, although about half of the labeled cells still resided in the WM and appeared immature. The first morphologically definable Bergmann glia, astrocytes, and oligodendrocytes were also observed. From 14 to 20 dpi, most labeled cells had developed into Bergmann glia, astrocytes, oligodendrocytes, and interneurons in their appropriate layers. When BAG injections were performed at P14, unipolar cells were initially observed, but the majority of these differentiated into myelinating oligodendrocytes in the WM and IGL by 17 dpi. Few immature cells were labeled by injections administered at P20, and these did not develop into mature glia, but into cells with lacy, fine processes, possible representing immature oligodendrocytes. In contrast, BAG-labeled progenitors of EGL produced only granule neurons. Thus, within the first 2 postnatal weeks, dividing progenitors in the WM migrate as immature cells into the cortex before differentiating into a variety of glia and interneurons. The genesis of oligodendrocytes continues through the 2nd postnatal week and largely ceases by P20. EGL cells do not produce glia, but only granule cells.

In this study we have investigated the electroencephalogram (EEG) background activity in patients with Alzheimer's disease (AD) using non-linear analysis methods. We calculated the Lempel-Ziv (LZ) complexity - applying two different sequence conversion methods - and the central tendency measure (CTM) of the EEG in 11 AD patients and 11 age-matched control subjects. CTM quantifies the degree of variability, while LZ complexity reflects the arising rate of new patterns along with the EEG time series. We did not find significant differences between AD patients and control subjects' EEGs with CTM. On the other hand, AD patients had significantly lower LZ complexity values (p<0.01) at electrodes P3 and O1 with a two-symbol sequence conversion, and P3, P4, O1 and T5 using three symbols. Our results show a decreased complexity of EEG patterns in AD patients. In addition, we obtained 90.9% sensitivity and 72.7% specificity at O1, and 72.7% sensitivity and 90.9% specificity at P3 and P4. These findings suggest that LZ complexity may contribute to increase the insight into brain dysfunction in AD in ways which are not possible with more classical and conventional statistical methods.

Electroencephalographic (EEG) data were recorded in 69 normal elderly (Nold), 88 mild cognitive impairment (MCI), and 109 mild Alzheimer's disease (AD) subjects at rest condition, to test whether the fronto-parietal coupling of EEG rhythms is in line with the hypothesis that MCI can be considered as a pre-clinical stage of the disease at group level. Functional coupling was estimated by synchronization likelihood of Laplacian-transformed EEG data at electrode pairs, which accounts for linear and non-linear components of that coupling. Cortical rhythms of interest were delta (2-4Hz), theta (4-8Hz), alpha 1 (8-10.5Hz), alpha 2 (10.5-13Hz), beta 1 (13-20Hz), beta 2 (20-30Hz), and gamma (30-40Hz). Compared to the Nold subjects, the AD patients presented a marked reduction of the synchronization likelihood (delta to gamma) at both fronto-parietal and inter-hemispherical (delta to beta 2) electrodes. As a main result, alpha 1 synchronization likelihood progressively decreased across Nold, MCI, and mild AD subjects at midline (Fz-Pz) and right (F4-P4) fronto-parietal electrodes. The same was true for the delta synchronization likelihood at right fronto-parietal electrodes (F4-P4). For these EEG bands, the synchronization likelihood correlated with global cognitive status as measured by the Mini Mental State Evaluation. The present results suggest that at group level, fronto-parietal coupling of the delta and alpha rhythms progressively becomes abnormal though MCI and mild AD. Future longitudinal research should evaluate whether the present EEG approach is able to predict the cognitive decline in individual MCI subjects.

The level of expertise of an operator may significantly influence his/her psychophysiological response to high task demand. A naive individual may invest considerable mental effort during performance of a difficult task and psychophysiological reactivity will be high compared to the psychophysiological response of a highly skilled operator. A study on multitasking performance was conducted to investigate the interaction between learning and task demand on psychophysiological reactivity. Thirty naive participants performed high and low demand versions of the Multi-attribute Task Battery (MATB) over a learning period of 64 min. High and low task demand setting were preset via a pilot study. Psychophysiological variables were collected from four channels of EEG (Cz, P3, P4, Pz), ECG, EOG and respiration rate to measure the impact of task demand and learning. Several variables were sensitive to the task demand manipulation but not time-on-task, e.g., heart rate, Theta activity at parietal sites. The sensitivity of certain variables to high demand was compromised by skill acquisition, e.g., respiration rate, suppression of alpha activity. A sustained learning effect was observed during the high demand condition only; multiple regression analyses revealed that specific psychophysiological variables predicted learning at different stages on the learning curve. The implications for the sensitivity of psychophysiological variables are discussed.

The purpose of the present investigation was to evaluate physical outcome after anterior cruciate ligament (ACL) reconstruction with early versus late initiation of open kinetic chain (OKC) exercises for the quadriceps in patients operated on either patellar tendon or hamstring grafts. Sixty-eight patients, 36 males and 32 females, with either patellar tendon graft (34 patients) or hamstring graft (34 patients) were enrolled in this study. All patients were randomly allocated to either early (the 4th postoperative week) or late (the 12th postoperative week) start of OKC exercises for the quadriceps, resulting in four subgroups: patellar tendon reconstruction, early start (P4) or late start (P12) of OKC quadriceps exercises, hamstring tendon reconstruction, early start (H4) or late start (H12) of quadriceps OKC exercises. Prior to surgery and 3, 5 and 7 months later, assessments of range of motion (goniometer), anterior knee laxity (KT-1000), postural sway (KAT 2000), thigh muscle torques (Kin-Com dynamometer) and anterior knee pain (anterior knee pain score) were evaluated. No significant group differences were found in terms of range of motion 3, 5 and 7 months postoperatively. The H4 group showed a significantly higher mean difference of laxity over time of 1.0 mm (CI: 0.18-1.86) than the P4 group (P=0.04). Within the same type of surgery, the H4 against the H12, the mean difference over time was 1.2 mm (0.37-2.1) higher in the H4 group than in the H12 group (P=0.01). There were no significant group differences in terms of postural sway or anterior knee pain at the different test occasions. Significant differences in trends (changes over time) were found when comparing the four groups, for both quadriceps muscle torques (P<0.001) and hamstring muscle torques (P<0.001). All groups, except the P4 group, reached preoperative values of quadriceps muscle torques at the 7 months follow-up. In the H4 and the H12 groups, significantly lower hamstring muscle torques at the 7 months follow-up compared with preoperative values were found. In conclusion, early start of OKC quadriceps exercises after hamstring ACL reconstruction resulted in significantly increased anterior knee laxity in comparison with both late start and with early and late start after bone-patellar tendon-bone ACL reconstruction. Furthermore, the early introduction of OKC exercises for quadriceps did not influence quadriceps muscle torques neither in patients operated on patellar tendon nor hamstring tendon grafts. On the contrary, it appears as if the choice of graft affected the strength of the specific muscle more than the type of exercises performed. Our results could not determine the appropriate time for starting OKC quadriceps exercises for patients who have undergone ACL reconstruction with hamstring tendon graft. Future studies of long-term results of anterior knee laxity and functional outcome are needed.

A greater incidence of temporomandibular joint (TMJ) pain is reported in females, suggesting that gonadal hormones may play a role in this condition. However, the exact roles of 17beta-estradiol (E2) and progesterone (P4) in TMJ pain are not completely known. Two experiments were performed to determine the separate roles of E2 and P4 in TMJ nociception at various stages of the estrous cycle. Ovariectomized (OVX) rats were cycled with physiological concentrations of E2 or P4. The E2-cycled rats then received bilateral TMJ injections of saline (SAL) or complete Freund's adjuvant (CFA) on the morning of diestrus-2 (low E2 condition) or proestrus (high E2 condition). As a control, OVX rats (no ovarian E2 and no replacement) were injected with SAL or CFA. The TMJ nociception was measured using a validated novel method in which an increase in meal duration directly correlated to the intensity of deep TMJ nociception. In the E2 experiment, CFA injection, but not SAL, increased TMJ nociception in the OVX group, but the effect was less pronounced in diestrus-2 and even less in proestrus. In the P4 experiment, the rats receiving TMJ CFA in diestrus-2 (end of minor P4 surge) did not show an increase in TMJ nociception, whereas the rats injected in proestrus (major P4 surge), estrus (low P4), and metestrus (low P4) had similar increases in TMJ nociception. The hormones' concentration did not affect TMJ IL-1beta, IL-6, C-C motif ligand 20, or C-X-C motif ligand 2 or the trigeminal ganglia calcitonin gene-related peptide. The high physiological concentrations of E2 observed at proestrus and the low P4 concentrations observed at diestrus-2 attenuated or eliminated CFA-induced TMJ nociception. The results suggest that the cyclic estrous cycle concentrations of E2 and P4 can influence CFA-induced TMJ nociception in the rat.

The orientation of proteins in ordered biological samples can be investigated using steady-state polarized fluorescence from probes conjugated to the protein. A general limitation of this approach is that the probes typically exhibit rapid orientational motion ("wobble") with respect to the protein backbone. Here we present a method for characterizing the extent of this wobble and for removing its effects from the available information about the static orientational distribution of the probes. The analysis depends on four assumptions: 1) the probe wobble is fast compared with the nanosecond time scale of its excited-state decay; 2) the orientational distributions of the absorption and emission transition dipole moments are cylindrically symmetrical about a common axis c fixed in the protein; 3) protein motions are negligible during the excited-state decay; 4) the distribution of c is cylindrically symmetrical about the director of the experimental sample. In a muscle fiber, the director is the fiber axis, F. All of the information on the orientational order of the probe that is available from measurements of linearly polarized fluorescence is contained in five independent polarized fluorescence intensities measured with excitation and emission polarizers parallel or perpendicular to F and with the propagation axis of the detected fluorescence parallel or perpendicular to that of the excitation. The analysis then yields the average second-rank and fourth-rank order parameters (and (P4)) of the angular distribution of c relative to F, andand, the average second-rank order parameters of the angular distribution for wobble of the absorption and emission transition dipole moments relative to c. The method can also be applied to other cylindrically ordered systems such as oriented lipid bilayer membranes and to processes slower than fluorescence that may be observed using longer-lived optically excited states.

IGF2 has been shown to be expressed preferentially from the paternally derived allele, although the maternal allele can be found active during both prenatal and postnatal development as well as in neoplastic tumours in humans. We addressed here whether or not the biallelic expression patterns that can be seen during postnatal human liver development reflected a coordinated change in the activities of the four promoters of human IGF2. We show here that the P2, P3 and P4 promoters, but not the P1 promoter, display monoallelic activity in embryonic, neonatal and younger infant liver specimens. The P2, P3 and P4 promoters can, however, be found active either monoallelically or biallelically or even monoallelically on opposite parental alleles in older infant and adult liver specimens. In contrast, H19, which is closely linked to IGF2, is monoallelically expressed in all postnatal liver samples analysed. We conclude that the functional imprinting status of IGF2 during postnatal liver development appears to be promoter/enhancer-specific and either partly or completely independent of H19.

We have examined regulation by protein kinase C (Ca2+/phospholipid-dependent enzyme) of thrombin-induced inositol polyphosphate accumulation in human platelets. When platelets are exposed to thrombin for 10 s, the protein kinase C inhibitor staurosporine causes inositol phosphate elevations over control values of 2.7-fold (inositol 1,4,5-trisphosphate (Ins(1,4,5)P3], 1.9-fold (inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4], and 1.2-fold (inositol 1,3,4-trisphosphate). In the same period, phosphatidic acid and diacylglycerol are unaffected. The myosin light chain kinase inhibitor ML-7 has no effect on inositol phosphate accumulations. Staurosporine does not inhibit Ins(1,4,5)P3 3-kinase and 5-phosphomonoesterase activities in saponin-permeabilized platelets incubated with exogenous Ins(1,4,5)P3 unless the platelets have been exposed to thrombin and protein kinase C is consequently activated. The protein kinase C agonist beta-phorbol 12,13-dibutyrate increases the Vmax of the 3-kinase 1.8-fold, with little effect on Km. Our results provide strong evidence for a role for protein kinase C in regulating inositol phosphate levels in thrombin-activated platelets. We propose that endogenously activated protein kinase C removes Ins(1,4,5)P3 by stimulating both 5-phosphomonoesterase and Ins(1,4,5)P3 3-kinase. Initial activation of phospholipase C does not appear to be affected by such protein kinase C. Inhibition of protein kinase C by staurosporine decreases 5-phosphomonoesterase activity. The resulting elevated Ins(1,4,5)P3, as substrate for Ins(1,4,5)P3 3-kinase, promotes production of Ins(1,3,4,5)P4, which also may accumulate through decreased 5-phosphomonoesterase activity and elevated Ca2+ levels. These factors apparently counteract the inhibitory effect on 3-kinase, yielding a net increase in Ins(1,3,4,5)P4.

AT-rich DNA elements play an important role in regulating cell-specific gene expression. One of the AT-rich DNA binding proteins, SATB1 is a novel type of transcription factor that regulates gene expression in the hematopoietic lineage through chromatin modification. Using DNA-affinity purification followed by mass spectrometry we identified and isolated a related protein, SATB2 from the developing rat cerebral cortex. SATB2 shows homology to SATB1 and the rat protein is practically identical to the mouse and human SATB2. Using competitive EMSA, we show that recombinant SATB2 protein binds with high affinity and specificity to AT-rich dsDNA. Using RT-PCR, Western analysis and immunohistochemistry we demonstrate that SATB2 expression is restricted to a subset of postmitotic, differentiating neurons in the rat neocortex at ages E16 and P4. We suggest that similar to its homologue SATB1, SATB2 is also involved in regulating gene expression through altering chromatin structure in differentiating cortical neurons.

This review summarizes recent findings on the non-genomic or membrane-initiated actions of progesterone that regulate the function of the normal and neoplastic mammalian ovary. This review focuses on three receptors: the classic progesterone receptor, the membrane progesterone receptors (MPRalpha, beta, and gamma) that were initially cloned from seatrout ovaries, and a progesterone binding protein referred to as progesterone receptor membrane component-1 (PGRMC1). Specifically, the structure of each of these receptors is compared and related to their capacity to activate various signal transduction pathways. Then the biological effects of P4 on the function of granulosa cells, luteal cells, ovarian surface epithelial cells, and ovarian cancers that are derived from the ovarian surface epithelial cells are discussed in relationship to the expression of each of these receptors. Whenever possible, studies involving human cells and tissues are presented, although data from other mammalian species are used to supplement the human studies to provide a more complete picture of this complex and rapidly developing area of membrane-initiated actions of progesterone.

The folate receptor (FR) type alpha is a promising target for diagnostic imaging agents and therapeutic intervention in major subtypes of gynecological malignancies; however, the receptor levels in the tumors are variable and are generally relatively low in estrogen receptor (ER)-positive tumors. Here we report that the FR-alpha gene promoter is repressed in the presence of 17beta-estradiol and derepressed by the antiestrogens tamoxifen and ICI 182780 in a promoter-specific and ER-alpha-dependent manner in carcinoma cell lines including HeLa (cervical carcinoma), BG-1 (ovarian carcinoma), and IGROV-1 (ovarian carcinoma). The ligand and ER dose response of the FR-alpha promoter and its time course paralleled those of a classical estrogen response element-mediated effect. Antiestrogens produced an ER-dependent increase of up to 36-fold in the expression of the endogenous FR-alpha gene. Deletion analysis and FR-alpha/SV40 promoter chimeras showed that the ER effect is mediated exclusively within the G/C-rich region in the TATA-less P4 promoter of FR-alpha; electrophoretic mobility shift analysis demonstrated interaction of ER at only one of three G/C-rich elements. ER-beta only modestly affected FR-alpha promoter activity but did not diminish the ER-alpha-mediated effects. The ER corepressor, SMRT, enhanced the repression by 17beta-estradiol/ER, but ER coactivators, including SRC family members, did not appreciably impact the ER ligand response. The results suggest that in ER+ tumors, FR-alpha expression is directly and actively suppressed and predict that a brief treatment with antiestrogens will boost FR-alpha expression by passive derepression, enhancing the efficacy of FR-targeted diagnostic and therapeutic applications. They also reveal novel aspects of gene repression by ER.

The features and regulation of uterine angiogenesis and vascular remodelling during pregnancy are poorly defined. Here we show that dynamic and variable decidual angiogenesis (sprouting, intussusception and networking), and active vigorous vascular remodelling such as enlargement and elongation of 'vascular sinus folding' (VSF) and mural cell drop-out occur distinctly in a spatiotemporal manner in the rapidly growing mouse uterus during early pregnancy - just after implantation but before placentation. Decidual angiogenesis is mainly regulated through VEGF-A secreted from the progesterone receptor (PR)-expressing decidual stromal cells which are largely distributed in the anti-mesometrial region (AMR). In comparison, P4 -PR-regulated VEGF-A-VEGFR2 signalling, ligand-independent VEGFR3 signalling and uterine natural killer (uNK) cells positively and coordinately regulate enlargement and elongation of VSF. During the postpartum period, Tie2 signalling could be involved in vascular maturation at the endometrium in a ligand-independent manner, with marked reduction of VEGF-A, VEGFR2 and PR expressions. Overall, we show that two key vascular growth factor receptors - VEGFR2 and Tie2 - strikingly but differentially regulate decidual angiogenesis and vascular remodelling in rapidly growing and regressing uteri in an organotypic manner.

BACKGROUND

Basal phenotype breast cancers (BPBC) are often associated with apparent epithelial to mesenchymal transition (EMT). The role of progesterone (P4) in regulating EMT of BPBC has not been reported.

METHODS

The EMT relevant biology was investigated in vitro using human BPBC cell models (MDA-MB468 and MDA-MB231) with P4, PR agonist (RU486), and PR antagonist (R5020) treatments. The essential role of membrane progesterone receptor alpha (mPRalpha) in the P4-regulated EMT was demonstrated by knocking down the endogenous gene and/or stably transfecting exogenous mPRalpha gene in the BPBC cell models.

RESULTS

The expression of snail and down-stream EMT proteins such as occludin, fibronectin, and E-cadherin was significantly regulated by P4 incubation, which was accompanied by cell morphological reversion from mesenchymal to epithelial phenotypes. In searching for the cell mediator of P4' action in the MDA-MB468 (MB468) cells, it was found that mPRalpha but not the nuclear PR has an essential role in the P4 mediated EMT inhibition. Knocking down the expression of mPRalpha with specific siRNA blocked the P4's effects on expression of the EMT proteins. In another BPBC cell line--MDA-MB231 (MB231), which is mPRalpha negative by Western blotting--P4 treatment did not alter cell proliferation and EMT protein expressions. Introduction of the exogenous mPRalpha cDNA into these cells caused cell proliferation, but not EMT, to become responsive to P4 treatment. In further studies, it was found that activation of the PI3K/Akt pathway is necessary for the P4-induced EMT reversion. To define the potential inter-mediate steps between mPRalpha and PI3K, we demonstrated that mPRalpha, caveolin-1 (Cav-1), and epidermal growth factor receptor (EGFR) are colocalized in the membrane of caveolar vesicle and the P4-repressed EMT in MB468 cells can be blocked by EGFR inhibitor (AG1478) and PI3K inhibitor (wortmannin).

CONCLUSIONS

Our data suggest that the signaling cascade of P4 induced mesenchymal repression is mediated through mPRalpha and other caveolae bound signaling molecules namely Cav-1, EGFR, and PI3K. This novel finding may have great impact on fully understanding the pathogenesis of BPBC and provide an essential clue for developing a targeted therapeutic strategy for treatment of BPBC.

P4-ATPases translocate aminophospholipids, such as phosphatidylserine (PS), to the cytosolic leaflet of membranes. PS is highly enriched in recycling endosomes (REs) and is essential for endosomal membrane traffic. Here, we show that PS flipping by an RE-localized P4-ATPase is required for the recruitment of the membrane fission protein EHD1. Depletion of ATP8A1 impaired the asymmetric transbilayer distribution of PS in REs, dissociated EHD1 from REs, and generated aberrant endosomal tubules that appear resistant to fission. EHD1 did not show membrane localization in cells defective in PS synthesis. ATP8A2, a tissue-specific ATP8A1 paralogue, is associated with a neurodegenerative disease (CAMRQ). ATP8A2, but not the disease-causative ATP8A2 mutant, rescued the endosomal defects in ATP8A1-depleted cells. Primary neurons from Atp8a2-/- mice showed a reduced level of transferrin receptors at the cell surface compared to Atp8a2+/+ mice. These findings demonstrate the role of P4-ATPase in membrane fission and give insight into the molecular basis of CAMRQ.

A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1-3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species.

Exosomes are thought to be released by all cells in the body and to be involved in intercellular communication. We tested whether neural exosomes can regulate the development of neural circuits. We show that exosome treatment increases proliferation in developing neural cultures and in vivo in dentate gyrus of P4 mouse brain. We compared the protein cargo and signaling bioactivity of exosomes released by hiPSC-derived neural cultures lacking MECP2, a model of the neurodevelopmental disorder Rett syndrome, with exosomes released by isogenic rescue control neural cultures. Quantitative proteomic analysis indicates that control exosomes contain multiple functional signaling networks known to be important for neuronal circuit development. Treating MECP2-knockdown human primary neural cultures with control exosomes rescues deficits in neuronal proliferation, differentiation, synaptogenesis, and synchronized firing, whereas exosomes from MECP2-deficient hiPSC neural cultures lack this capability. These data indicate that exosomes carry signaling information required to regulate neural circuit development.

Cation-mediated RNA folding from extended to compact, biologically active conformations relies on a temporal balance of forces. The Mg2 +-mediated folding of the Tetrahymena thermophila ribozyme is characterized by rapid nonspecific collapse followed by tertiary-contact-induced compaction. This article focuses on an autonomously folding portion of the Tetrahymena ribozyme, its P4-P6 domain, in order to probe one facet of the rapid collapse: chain flexibility. The time evolution of P4-P6 folding was followed by global and local measures as a function of Mg2 + concentration. While all concentrations of Mg2 + studied are sufficient to screen the charge on the helices, the rates of compaction and tertiary contact formation diverge as the concentration of Mg2 + increases; collapse is greatly accelerated by Mg2 +, while tertiary contact formation is not. These studies highlight the importance of chain stiffness to RNA folding; at 10 mM Mg2 +, a stiff hinge limits the rate of P4-P6 folding. At higher magnesium concentrations, the rate-limiting step shifts from hinge bending to tertiary contact formati

We have sequenced the control region of the uvrC protein including two open reading frames (ORF) encoding polypeptides of 28 kd and 23 kd molecular weight. The uvrC gene is preceded by five promoters. The P1, P2a and P2b promoter sequences are 5' to the 28 kd and the 23 kd proteins respectively. The P3 and P4 promoters are located within the structural gene for the 23 kd protein. The P3 promoter is required for adequate in vivo expression. There are three putative lexA protein binding sites, detected at the 3' end of the 28 kd protein (lexA1), within the coding sequences for the 23 kd protein (lexA2) and within the P3 promoter (lexA3). Promoter P2 is responsible for transcription of the uvrC gene, producing transcripts of 2.8 and 1.6 kb. The upstream region including the 28 kd protein is required for enhanced expression under non-induced conditions. These results show that the uvrC gene is controlled by multiple promoters and is transcribed as part of a multigene unit.

The time-course of monovalent cation-induced folding of the L-21 Sca1 Tetrahymena thermophila ribozyme and a selected mutant was quantitatively followed using synchrotron X-ray (.OH) footprinting. Initiating folding by increasing the concentration of either Na+ or K+ to 1.5M from an initial condition of approximately 0.008 M Na+ at 42 degrees C resulted in the complete formation of tertiary contacts within the P5abc subdomain and between the peripheral helices within the dead time of our measurements (k>50 s(-1)). These results contrast with folding rates of 2-0.2 s(-1) previously observed for formation of these contacts in 10mM Mg2+ from the same initial condition. Thus, the initial formation of native tertiary contacts is inhibited by divalent but not monovalent cations. The native contacts within the catalytic core form without a detectable burst phase at rates of 0.4-1.0 s(-1) in a manner reminiscent of the Mg2+-dependent folding behavior, although tenfold faster. The tertiary interactions stabilizing the catalytic core interaction with P4-P6 and P2.1, as well as one of the protections internal for the P4-P6 domain, display progress curves with appreciable burst amplitudes and a phase comparable in rate to that of the catalytic core. That the slow folding of the ribozyme's core is a consequence of the alt-P3 secondary structure is shown by the 100% burst phase amplitudes that are observed for folding of the U273A mutant ribozyme within which the native secondary structure (P3) is strengthened. Thus, formation of a misfolded intermediate(s) resulting from the alt-P3 secondary structure is independent of ion valency while the rate at which the respective intermediates are resolved is sensitive to ion valency. The overall portrait painted by these results is that ion valency differentially affects steps in the folding process and that folding in monovalent ion alone for the U273A mutant Tetrahymena ribozyme is fast and direct.