Short-term homeostatic visual neuroplasticity in adolescents after two hours of monocular deprivation.

In healthy adults with normal vision, temporary deprivation of one eye's visual experience produces transient yet robust homeostatic plasticity effects, where the deprived eye becomes more dominant. This shift in ocular dominance is short-lived and compensatory. Previous work shows that monocular deprivation decreases resting state gamma aminobutyric acid (GABA; inhibitory neurotransmitter) levels in visual cortex, and that those with the greatest reduction in GABA have stronger shifts due to monocular deprivation. Components of the GABAergic system in visual cortex vary with age (early childhood, early teen years, ageing); hence if GABA is critical to homeostatic plasticity within the visual system, adolescence may be a key developmental period where differences in plasticity manifest. Here we measured short-term visual deprivation effects on binocular rivalry in 24 adolescents (aged 10-15 years) and 23 young adults (aged 20-25 years). Despite differences in baseline features of binocular rivalry (adolescents showed more mixed percept p < 0.001 and a tendency for faster switching p = 0.06 compared to adults), deprived eye dominance increased (p = 0.01) similarly for adolescents and adults after two hours of patching. Other aspects of binocular rivalry - time to first switch (heralding the onset of rivalry) and mixed percept - were unaltered by patching. These findings suggest that binocular rivalry after patching can be used as a behavioral proxy for experience-dependent visual cortical plasticity in adolescents in the same way as adults, and that homeostatic plasticity to compensate for temporarily reduced visual input is established and effective by adolescence.

UBXN2A suppresses the Rictor-mTORC2 signaling pathway, an established tumorigenic pathway in human colorectal cancer.

The mTORC2 pathway plays a critical role in promoting tumor progression in human colorectal cancer (CRC). The regulatory mechanisms for this signaling pathway are only partially understood. We previously identified UBXN2A as a novel tumor suppressor protein in CRCs and hypothesized that UBXN2A suppresses the mTORC2 pathway, thereby inhibiting CRC growth and metastasis. We first used murine models to show that haploinsufficiency of UBXN2A significantly increases colon tumorigenesis. Induction of UBXN2A reduces AKT phosphorylation downstream of the mTORC2 pathway, which is essential for a plethora of cellular processes, including cell migration. Meanwhile, mTORC1 activities remain unchanged in the presence of UBXN2A. Mechanistic studies revealed that UBXN2A targets Rictor protein, a key component of the mTORC2 complex, for 26S proteasomal degradation. A set of genetic, pharmacological, and rescue experiments showed that UBXN2A regulates cell proliferation, apoptosis, migration, and colon cancer stem cells (CSCs) in CRC. CRC patients with a high level of UBXN2A have significantly better survival, and high-grade CRC tissues exhibit decreased UBXN2A protein expression. A high level of UBXN2A in patient-derived xenografts and tumor organoids decreases Rictor protein and suppresses the mTORC2 pathway. These findings provide new insights into the functions of an ubiquitin-like protein by inhibiting a dominant oncogenic pathway in CRC.

UCHL1 Regulates Lipid and Perilipin 2 Level in Skeletal Muscle.

Ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that was originally found in neurons. We found that UCHL1 is highly expressed in slow oxidative skeletal muscles, but its functions remain to be fully understood. In this study, we observed that UCHL1 protein levels in skeletal muscle and C2C12 myotubes were downregulated by fasting or glucose starvation respectively. Skeletal muscle selective knockout (smKO) of UCHL1 resulted in a significant reduction of lipid content in skeletal muscle and improved glucose tolerance. UCHL1 smKO did not significantly change the levels of key proteins involved in oxidative metabolism such as SDHA, Akt, or PDH. Interestingly, while the levels of the major lipases and lipid transporters were unchanged, perilipin 2 was significantly downregulated in UCHL1 smKO muscle. Consistently, in C2C12 myotubes, UCHL1 siRNA knockdown also reduced perilipin 2 protein level. This data suggests that UCHL1 may stabilize perilipin 2 and thus lipid storage in skeletal muscle.

Developing a Screening Tool for Areas of Abnormal Central Vision Using Visual Stimuli With Natural Scene Statistics.

PURPOSE: Previous studies show that some visual field (VF) defects are detectable from visual search behavior; for example, when watching video. Here, we developed and tested a VF testing approach that measures the number of fixations to find targets on a background with spatial frequency content similar to natural scenes. METHODS: Twenty-one older controls and 20 people with glaucoma participated. Participants searched for a Gabor (6 c/°) that appeared in one of 25 possible locations within a 15° (visual angle) 1/f noise background (RMS contrast: 0.20). Procedure performance was assessed by calculating sensitivity and specificity for different combinations of control performance limits (p = 95%, 98%, 99%), number of target locations with fixations outside control performance limits (k = 0 to 25) and number of repeated target presentations (n = 1 to 20). RESULTS: Controls made a median of two to three fixations (twenty-fifth to seventy-fifth percentile: two to four) to locate the target depending on location. A VF was flagged "abnormal" when the number of fixations was greater than the p = 99% for k = 3 or more locations with n = 2 repeated presentations, giving 85% sensitivity and 95.2% specificity. The median test time for controls was 85.71 (twenty-fifth to seventy-fifth percentile: 66.49-113.53) seconds. CONCLUSION: Our prototype test demonstrated effective and efficient screening of abnormal areas in central vision. TRANSLATIONAL RELEVANCE: Visual search behavior can be used to detect central vision loss and may produce results that relate well to performance in natural visual environments.

Enhanced pro-BDNF-p75NTR pathway activity in denervated skeletal muscle.

AIMS: Brain derived neurotrophic factor (BDNF) and the related receptors TrkB and p75NTR are expressed in skeletal muscle, yet their functions remain to be fully understood. Skeletal muscle denervation, which occurs in spinal injury, peripheral neuropathies, and aging, negatively affects muscle mass and function. In this study, we wanted to understand the role of BDNF, TrkB, and p75NTR in denervation-induced adverse effects on skeletal muscle. MAIN METHODS: Mice with unilateral sciatic denervation were used. Protein levels of pro- and mature BDNF, TrkB, p75NTR, activations of their downstream signaling pathways, and inflammation in the control and denervated muscle were measured with Western blot and tissue staining. Treatment with a p75NTR inhibitor and BDNF skeletal muscle specific knockout in mice were used to examine the role of p75NTR and pro-BDNF. KEY FINDINGS: In denervated muscle, pro-BDNF and p75NTR were significantly upregulated, and JNK and NF-kB, two major downstream signaling pathways of p75NTR, were activated, along with muscle atrophy and inflammation. Inhibition of p75NTR using LM11A-31 significantly reduced JNK activation and inflammatory cytokines in the denervated muscle. Moreover, skeletal muscle specific knockout of BDNF reduced pro-BDNF level, JNK activation and inflammation in the denervated muscle. SIGNIFICANCE: These results reveal for the first time that the upregulation of pro-BDNF and activation of p75NTR pathway are involved in denervation-induced inflammation in skeletal muscle. The results suggest that inhibition of pro-BDNF-p75NTR pathway can be a new target to treat skeletal muscle inflammation.

Contrast Sensitivity on 1/f Noise Is More Greatly Impacted by Older Age for the Fovea Than Parafovea.

SIGNIFICANCE: Contrast sensitivity changes across the visual field with age and is often measured clinically with various forms of perimetry on plain backgrounds. In daily life, the visual scene is more complicated, and therefore, the standard clinical measures of contrast sensitivity may not predict a patient's visual experience in more natural environments. PURPOSE: This study aims to determine whether contrast thresholds in older adults are different from younger adults when measured on a 1/f noise background (a nonuniform background whose spatial frequency content is similar to those present in the natural vision environments). METHODS: Twenty younger (age range, 20 to 35 years) and 20 older adults (age range, 61 to 79 years) with normal ocular health were recruited. Contrast thresholds were measured for a Gabor patch of 6 cycles per degree (sine wave grating masked by a Gaussian envelope of standard deviation 0.17°) presented on 1/f noise background (root-mean-square contrast, 0.05 and 0.20) that subtended 15° diameter of the central visual field. The stimulus was presented at four eccentricities (0°, 2°, 4°, and 6°) along the 45° meridian in the noise background, and nine contrast levels were tested at each eccentricity. The proportion of correct responses for detecting the target at each eccentricity was obtained, and psychometric functions were fit to estimate the contrast threshold. RESULTS: Older adults demonstrate increased contrast thresholds compared with younger adults. There was an eccentricity-dependent interaction with age, with the difference between groups being highest in the fovea compared with other eccentricities. Performance was similar for the two noise backgrounds tested. CONCLUSIONS: Our results revealed a strong eccentricity dependence in performance between older and younger adults, highlighting age-related differences in the contrast detection mechanisms between fovea and parafovea for stimuli presented on nonuniform backgrounds.

SNAIL Transctiption factor in prostate cancer cells promotes neurite outgrowth.

Neurite outgrowth involves reciprocal signaling interactions between tumor cells and nerves where invading tumor cells have acquired the ability to respond to pro-invasive signals within the nerve environment. Neurite outgrowth could serve as a mechanism leading to invasion of cancer cells into the nerve sheath and subsequent metastasis. Snail transcription factor can promote migration and invasion of prostate cancer cells. We hypothesized that prostate cancer cell interaction with nerve cells will be mediated by Snail expression within prostate cancer cells. For this study we utilized various prostate cancer cell lines: C4-2 non-silencing (NS, control); C4-2 Snail shRNA, (stable Snail knockdown); LNCaP Neo (empty vector control) and LNCaP Snail (stably over-expressing Snail). Cancer cell adhesion and migration towards nerve cells (snF96.2 or NS20Y) was examined by co-culture assays. Conditioned media (CM) collected from C4-2 cells was cultured with nerve cells (PC-12 or NS20Y) for 48 h followed by qualitative or quantitative neurite outgrowth assay. Our results showed that cancer cells expressing high levels of Snail (LNCaP Snail/C4-2 NS) displayed significantly higher migration adherence to nerve cells, compared to cells with lower levels of Snail (LNCaP Neo/C4-2 Snail shRNA). Additionally, LNCaP Snail or C4-2 NS (Snail-high) CM led to a higher neurite outgrowth compared to the LNCaP Neo or C4-2 Snail shRNA (Snail-low). In conclusion, Snail promotes migration and adhesion to nerve cells, as well as neurite outgrowth via secretion of soluble factors. Therefore, targeting cancer cell interaction with nerves may contribute to halting prostate cancer progression/metastasis.

UCHL1 regulates oxidative activity in skeletal muscle.

Ubiquitin C-terminal Hydrolase L1 (UCHL1) is a deubiquitinating enzyme that was originally identified in neurons. Our recent study showed that UCHL1 was expressed in C2C12 myoblast cells and mouse skeletal muscle. Here we report that in mouse skeletal muscle, UCHL1 is primarily expressed in oxidative muscle fibers. Skeletal muscle specific gene knockout (smKO) of UCHL1 in mice reduced oxidative activity in skeletal muscle measured by SDH staining. The in situ muscle contraction test revealed that gastrocnemius muscle from UCHL1 smKO mice was more prone to fatigue in response to the repetitive stimulation. This data suggests that UCHL1 plays a role in maintenance of muscle oxidative metabolism. Moreover, UCHL1 smKO caused a significant reduction in key proteins that are involved in mitochondrial oxidative phosphorylation in soleus muscles, suggesting that UCHL1 may be involved in regulation of mitochondrial content and function. Immunostaining showed the co-localization of UCHL1 and mitochondrial marker VDAC in skeletal muscle. Mitochondrial fractionation assay revealed that, although UCHL1 was primarily present in the cytosolic fraction, a low level of UCHL1 protein was present in mitochondrial fraction. The level of phosphorylation of AMPKα, a master regulator of mitochondrial biogenesis, were unchanged in UCHL1 smKO muscle. On the other hand, immunoprecipitation from soleus muscle sample indicated the interaction between UCHL1 and HSP60, a chaperon protein that is involved in mitochondrial protein transport. There was a trend of downregulation of HSP60 in UCHL1 smKO muscle. Overall, our data suggests UCHL1 is a novel regulator of mitochondrial function and oxidative activity in skeletal muscle.

The Capacity of High-Grade Serous Ovarian Cancer Cells to Form Multicellular Structures Spontaneously along Disease Progression Correlates with Their Orthotopic Tumorigenicity in Immunosuppressed Mice.

Many studies have examined the biology, genetics, and chemotherapeutic response of ovarian cancer's solid component; its liquid facet, however, remains critically underinvestigated. Floating within peritoneal effusions known as ascites, ovarian cancer cells form multicellular structures, creating a cancer niche in suspension. This study explores the pathobiology of spontaneously formed, multicellular, ovarian cancer structures derived from serous ovarian cancer cells isolated along disease evolution. It also tests their capacity to cause peritoneal disease in immunosuppressed mice. Results stem from an analysis of cell lines representing the most frequently diagnosed ovarian cancer histotype (high-grade serous ovarian cancer), derived from ascites of the same patient at distinct stages of disease progression. When cultured under adherent conditions, in addition to forming cellular monolayers, the cultures developed areas in which the cells grew upwards, forming densely packed multilayers that ultimately detached from the bottom of the plates and lived as free-floating, multicellular structures. The capacity to form foci and to develop multicellular structures was proportional to disease progression at the time of ascites extraction. Self-assembled in culture, these structures varied in size, were either compact or hollow, irregular, or spheroidal, and exhibited replicative capacity and an epithelial nature. Furthermore, they fully recreated ovarian cancer disease in immunosuppressed mice: accumulation of malignant ascites and pleural effusions; formation of discrete, solid, macroscopic, peritoneal tumors; and microscopic growths in abdominal organs. They also reproduced the histopathological features characteristic of high-grade serous ovarian cancer when diagnosed in patients. The following results encourage the development of therapeutic interventions to interrupt the formation and/or survival of multicellular structures that constitute a floating niche in the peritoneal fluid, which in turn halts disease progression and prevents recurrence.

INTRAOCULAR PRESSURE CHANGES AFTER DEXAMETHASONE IMPLANT IN PATIENTS WITH GLAUCOMA AND STEROID RESPONDERS.

PURPOSE: To assess the changes in intraocular pressure (IOP) after dexamethasone (DEX) implant in patients with glaucoma or history of steroid responders. METHODS: A retrospective study of patients who received DEX implant was conducted in a tertiary care center in India. Demographic details and IOP measurements at preinjection and postinjection were collected. The proportion of patients with and without IOP rise after DEX implant was noted, and the number of antiglaucoma medications to control the IOP was analyzed. The changes in IOP were also compared in the group with no glaucoma/steroid responder. RESULTS: A total of 815 patients, 767 patients in the nonglaucoma group and 48 patients in the glaucoma referral group, who underwent DEX implant were included in this study. The overall mean (SD) age of study participants was 56.3 (SD = 12.6) years. The overall mean (SD) IOP at baseline and at follow-up after injection for both nonglaucoma and glaucoma referral groups was found to be significant (P < 0.001). The differences in IOP measurements across follow-ups after DEX implant were found to be significant in both nonglaucoma (P < 0.001) and glaucoma referral groups (P < 0.001). Among the study patients in the IOP-rise group, 46.43% had maximum IOP rise in 1-week follow-up and 39% in 2-week follow-up, where 78.6% showed IOP controlled with 1 antiglaucoma medication. CONCLUSION: The rise in IOP after DEX implant was noted within first 2 weeks, which can be managed with topical antiglaucoma medications. Hence, with a close early follow-up monitoring of IOP, and timely medical intervention, DEX implant can be performed in patients with glaucoma.

UBXN2A enhances CHIP-mediated proteasomal degradation of oncoprotein mortalin-2 in cancer cells.

Overexpression of oncoproteins is a major cause of treatment failure using current chemotherapeutic drugs. Drug-induced degradation of oncoproteins is feasible and can improve clinical outcomes in diverse types of cancers. Mortalin-2 (mot-2) is a dominant oncoprotein in several tumors, including colorectal cancer (CRC). In addition to inactivating the p53 tumor suppressor protein, mot-2 enhances tumor cell invasion and migration. Thus, mot-2 is considered a potential therapeutic target in several cancer types. The current study investigated the biological role of a ubiquitin-like protein called UBXN2A in the regulation of mot-2 turnover. An orthogonal ubiquitin transfer technology followed by immunoprecipitation, in vitro ubiquitination, and Magnetic Beads TUBE2 pull-down experiments revealed that UBXN2A promotes carboxyl terminus of the HSP70-interacting protein (CHIP)-dependent ubiquitination of mot-2. We subsequently showed that UBXN2A increases proteasomal degradation of mot-2. A subcellular compartmentalization experiment revealed that induced UBXN2A decreases the level of mot-2 and its chaperone partner, HSP60. Pharmacological upregulation of UBXN2A using a small molecule, veratridine (VTD), decreases the level of mot-2 in cancer cells. Consistent with the in vitro results, UBXN2A+/- mice exhibited selective elevation of mot-2 in colon tissues. An in vitro Anti-K48 TUBE isolation approach showed that recombinant UBXN2A enhances proteasomal degradation of mot-2 in mouse colon tissues. Finally, we observed enhanced association of CHIP with the UBXN2A-mot-2 complex in tumors in an azoxymethane/dextran sulfate sodium-induced mouse CRC model. The existence of a multiprotein complex containing UBXN2A, CHIP, and mot-2 suggests a synergistic tumor suppressor activity of UBXN2A and CHIP in mot-2-enriched tumors. This finding validates the UBXN2A-CHIP axis as a novel and potential therapeutic target in CRC.

Development of a Proximity Labeling System to Map the Chlamydia trachomatis Inclusion Membrane.

Chlamydia grows within a membrane-bound vacuole termed an inclusion. The cellular processes that support the biogenesis and integrity of this pathogen-specified parasitic organelle are not understood. Chlamydia secretes integral membrane proteins called Incs that insert into the chlamydial inclusion membrane (IM). Incs contain at least two hydrophobic transmembrane domains flanked by termini, which vary in size and are exposed to the host cytosol. In addition, Incs are temporally expressed during the chlamydial developmental cycle. Data examining Inc function are limited because of (i) the difficulty in working with hydrophobic proteins and (ii) the inherent fragility of the IM. We hypothesize that Incs function collaboratively to maintain the integrity of the chlamydial inclusion with small Incs organizing the IM and larger Incs interfacing with host cell machinery. To study this hypothesis, we have adapted a proximity-labeling strategy using APEX2, a mutant soybean ascorbate peroxidase that biotinylates interacting and proximal proteins within minutes in the presence of H2O2 and its exogenous substrate, biotin-phenol. We successfully expressed, from an inducible background, APEX2 alone, or fusion proteins of IncATM (TM = transmembrane domain only), IncA, and IncF with APEX2 in Chlamydia trachomatis serovar L2. IncF-APEX2, IncA TM -APEX2, and IncA-APEX2 localized to the IM whereas APEX2, lacking a secretion signal, remained associated with the bacteria. We determined the impact of overexpression on inclusion diameter, plasmid stability, and Golgi-derived sphingomyelin acquisition. While there was an overall impact of inducing construct expression, IncF-APEX2 overexpression most negatively impacted these measurements. Importantly, Inc-APEX2 expression in the presence of biotin-phenol resulted in biotinylation of the IM. These data suggest that Inc expression is regulated to control optimal IM biogenesis. We subsequently defined lysis conditions that solubilized known Incs and were compatible with pulldown conditions. Importantly, we have created powerful tools to allow direct examination of the dynamic composition of the IM, which will provide novel insights into key interactions that promote chlamydial growth and development within the inclusion.

Mifepristone increases mRNA translation rate, triggers the unfolded protein response, increases autophagic flux, and kills ovarian cancer cells in combination with proteasome or lysosome inhibitors.

The synthetic steroid mifepristone blocks the growth of ovarian cancer cells, yet the mechanism driving such effect is not entirely understood. Unbiased genomic and proteomic screenings using ovarian cancer cell lines of different genetic backgrounds and sensitivities to platinum led to the identification of two key genes upregulated by mifepristone and involved in the unfolded protein response (UPR): the master chaperone of the endoplasmic reticulum (ER), glucose regulated protein (GRP) of 78 kDa, and the CCAAT/enhancer binding protein homologous transcription factor (CHOP). GRP78 and CHOP were upregulated by mifepristone in ovarian cancer cells regardless of p53 status and platinum sensitivity. Further studies revealed that the three UPR-associated pathways, PERK, IRE1α, and ATF6, were activated by mifepristone. Also, the synthetic steroid acutely increased mRNA translation rate, which, if prevented, abrogated the splicing of XBP1 mRNA, a non-translatable readout of IRE1α activation. Moreover, mifepristone increased LC3-II levels due to increased autophagic flux. When the autophagic-lysosomal pathway was inhibited with chloroquine, mifepristone was lethal to the cells. Lastly, doses of proteasome inhibitors that are inadequate to block the activity of the proteasomes, caused cell death when combined with mifepristone; this phenotype was accompanied by accumulation of poly-ubiquitinated proteins denoting proteasome inhibition. The stimulation by mifepristone of ER stress and autophagic flux offers a therapeutic opportunity for utilizing this compound to sensitize ovarian cancer cells to proteasome or lysosome inhibitors.

In vitro and in vivo platelet targeting by cyclic RGD-modified liposomes.

Cell-selective delivery using ligand-decorated nanoparticles is a promising modality for treating cancer and vascular diseases. We are developing liposome nanoparticles surface-modified by RGD peptide ligands having targeting specificity to integrin GPIIb-IIIa. This integrin is upregulated and stimulated into a ligand-binding conformation on the surface activated platelets. Activated-platelet adhesion and aggregation are primary events in atherosclerosois, thrombosis, and restenosis. Hence, platelet-targeted nanoparticles hold the promise of vascular site-selective delivery of drugs and imaging probes. Here, we report in vitro and ex vivo microscopy studies of platelet-targeting by liposomes surface-modified with a cyclic RGD peptide. The peptide-modified liposomes were labeled either with a lipophilic fluorophore or with lipid-tethered Nanogold(R). For in vitro tests, coverslip-adhered activated human platelets were incubated with probe-labeled liposomes, followed by analysis with fluorescence microscopy, phase contrast microscopy, and scanning electron microscopy (SEM). For in vivo tests, the liposomes were introduced within a catheter-injured carotid artery restenosis model in rats and post-euthanasia, the artery was imaged ex vivo by fluorescence microscopy and SEM. All microscopy results showed successful platelet-targeting by the peptide-modified liposomes. The in vitro SEM results also enabled visualization of nanoscopic liposomes attached to activated platelets. The results validate our nanoparticle design for site-selective vascular delivery.

Affinity manipulation of surface-conjugated RGD peptide to modulate binding of liposomes to activated platelets.

Platelet adhesion, activation and fibrinogen-mediated aggregation are primary events in vascular thrombosis and occlusion. An injectable delivery system that can carry thrombolytics selectively to the sites of active platelet aggregation has immense potential in minimally invasive targeted therapy of vascular occlusion. To this end we are studying liposomes surface-modified by fibrinogen-mimetic RGD motifs that can selectively target and bind integrin GPIIb-IIIa on activated platelets. Here we report liposome surface-modification with a conformationally constrained high affinity cyclic RGD motif to modulate the GPIIb-IIIa-binding capability of the liposomes. Such affinity enhancement is important for practical in vivo applications to compete with native fibrinogen towards binding GPIIb-IIIa. The platelet-binding of RGD-modified liposomes was studied by fluorescence and scanning electron microscopy, and flow cytometry, in vitro. Binding of RGD-modified liposomes was also tested in vivo in a rat carotid injury model and analyzed ex vivo by fluorescence microscopy. The results from all experiments show that cyclic RGD-liposomes bind activated platelets significantly higher compared to linear RGD-liposomes. Hence, the results establish the feasibility of modulating the platelet-targeting and binding ability of vascularly targeted liposomes by manipulating the affinity of surface-modifying ligands.

Cap-independent translation through the p27 5'-UTR.

Several recent publications have explored cap-independent translation through an internal ribosome entry site (IRES) in the 5'-UTR of the mRNA encoding the cyclin-dependent kinase inhibitor p27. The major experimental tool used in these reports was the use of bicistronic reporter constructs in which the 5'-UTR was inserted between the upstream and downstream cistrons. None of these reports has completely ruled out the possibility that the 5'-UTR has either cryptic promoter activity or a cryptic splice acceptor site. Either of these possibilities could result in expression of a monocistronic mRNA encoding the downstream cistron and false identification of an IRES. Indeed, Liu et al. recently published data suggesting that the p27 5'-UTR harbors cryptic promoter activity which accounts for its putative IRES activity. In this report, we have explored this potential problem further using promoterless bicistronic constructs coupled with RNase protection assays, siRNA knockdown of individual cistrons, RT-PCR to detect mRNA encoded by the bicistronic reporter with high sensitivity, direct transfection of bicistronic mRNAs, and insertion of an iron response element into the bicistronic reporter. The results do not support the conclusion that the p27 5'-UTR has significant functional promoter activity or cryptic splice sites, but rather that it is able to support cap-independent initiation of translation.

ABri peptide associated with familial British dementia forms annular and ring-like protofibrillar structures.

Amyloid plaque deposition involves the aggregation of normally soluble proteins into insoluble amyloid fibrils (fibrillization) and proceeds through intermediates with distinct morphologies, including spherical aggregates, protofibrils, and mature fibrils. Recently, a novel annular protofibril-like intermediate with unique pore-like properties was produced by alpha-synuclein, A beta-Arctic and amylin, which are proteins associated with Parkinson's disease, Alzheimer's disease, and type-II diabetes. The observation of annular structures coupled with size selective channel-like activity by these proteins suggests that these structures may be responsible for vesicle permeability by ion-channel formation. Using atomic force spectroscopy, we report here that the ABri peptide associated with familial British dementia produces similar annular and ring-like protofibril structures during the following sequence of events: spherical aggregates (0.4-1.5 nm height)-->chain-like protofibrils (1.5-2.3 nm height)-->ring-like protofibrils and annular protofibrils (1.5-2.3 nm height). This suggests that ABri fibrillization occurs in a similar fashion to other amyloidogenic proteins and that the annular protofibrillar structures may represent a common amyloid intermediate.

pH-dependent amyloid and protofibril formation by the ABri peptide of familial British dementia.

The ABri is a 34 residue peptide that is the major component of amyloid deposits in familial British dementia. In the amyloid deposits, the ABri peptide adopts aggregated beta-pleated sheet structures, similar to those formed by the Abeta peptide of Alzheimer's disease and other amyloid forming proteins. As a first step toward elucidating the molecular mechanisms of the beta-amyloidosis, we explored the ability of the environmental variables (pH and peptide concentration) to promote beta-sheet fibril structures for synthetic ABri peptides. The secondary structures and fibril morphology were characterized in parallel using circular dichroism, atomic force microscopy, negative stain electron microscopy, Congo red, and thioflavin-T fluorescence spectroscopic techniques. As seen with other amyloid proteins, the ABri fibrils had characteristic binding with Congo red and thioflavin-T, and the relative amounts of beta-sheet and amyloid fibril-like structures are influenced strongly by pH. In the acidic pH range 3.1-4.3, the ABri peptide adopts almost exclusively random structure and a predominantly monomeric aggregation state, on the basis of analytical ultracentrifugation measurements. At neutral pH, 7.1-7.3, the ABri peptide had limited solubility and produced spherical and amorphous aggregates with predominantly beta-sheet secondary structure, whereas at slightly acidic pH, 4.9, spherical aggregates, intermediate-sized protofibrils, and larger-sized mature amyloid fibrils were detected by atomic force microscopy. With aging at pH 4.9, the protofibrils underwent further association and eventually formed mature fibrils. The presence of small amounts of aggregated peptide material or seeds encourage fibril formation at neutral pH, suggesting that generation of such seeds in vivo could promote amyloid formation. At slightly basic pH, 9.0, scrambling of the Cys5-Cys22 disulfide bond occurred, which could lead to the formation of covalently linked aggregates. The presence of the protofibrils and the enhanced aggregation at slightly acidic pH is consistent with the behavior of other amyloid-forming proteins, which supports the premise that a common mechanism may be involved in protein misfolding and beta-amyloidosis.

p27(Kip1) enhances myelin basic protein gene promoter activity.

The process of oligodendrocyte differentiation is a complex event that requires cell cycle withdrawal, followed by the activation of a specific transcriptional program responsible for the synthesis of myelin genes. Because growth arrest precedes differentiation, we sought to investigate the role of cell cycle molecules in the activation of myelin gene promoters. We hypothesized that the cell cycle inhibitor p27(Kip1), which is primarily responsible for arresting proliferating oligodendrocyte progenitors, may be involved in the transcriptional regulation of myelin genes. In agreement with this hypothesis, overexpression of p27(Kip1) in the CG4 cell line, but not in 3T3 fibroblasts, enhances the expression of luciferase driven by the myelin basic protein (MBP) promoter. Interestingly, this effect is specific for p27(Kip1); overexpression of other cell cycle inhibitors had no effect. Additionally, this effect is independent of halting the cell cycle; treatment with the cell cycle blocker roscovitine did not affect MBP promoter usage. We conclude that p27(Kip1) contributes to oligodendrocyte differentiation by regulating transcription of the MBP gene.