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Hydration of denatured and molten globule proteins.

The hydration of nonnative states is central to protein folding and stability but has been probed mainly by indirect methods. Here we use water 17O relaxation dispersion to monitor directly the internal and external hydration of alpha-lactalbumin, lysozyme, ribonuclease A, apomyoglobin and carbonic anhydrase in native and nonnative states. The results show that nonnative proteins are more structured and less solvent exposed than commonly believed. Molten globule proteins preserve most of the native internal hydration sites and have native-like surface hydration. Proteins denatured by guanidinium chloride are not fully solvent exposed but contain strongly perturbed occluded water. These findings shed new light on hydrophobic stabilization of proteins.

Animals↗

Transient YAP activation uncovers the neurogenic potential of proliferative mammalian Müller glia.

The Hippo pathway effector YAP promotes spontaneous proliferation of Müller glia (MG), suggesting that bypassing Hippo signaling and activating YAP could enhance retinal regeneration. However, whether proliferative adult MGs retain meaningful neurogenic competence remains unclear. Here, using viral delivery of a Hippo-resistant YAP variant to wild-type adult MGs, we achieved transient YAP activation in adult MGs, inducing proliferation followed by cell-cycle withdrawal and differentiation. Intersectional genetic lineage tracing and EdU labeling, combined with transcriptomic analyses, revealed that YAP-activated MGs predominantly regenerate MGs, whereas only a subset gives rise to bipolar cell-like neurons. These results indicate that proliferative MGs acquire a state resembling that of late-stage retinal progenitors, with limited neurogenic lineage potential. We conclude that YAP-activated cell-cycle reentry inefficiently reprograms adult MGs toward photoreceptor or ganglion cell fates. These findings define the limited competence of proliferative adult MGs to contribute to neurogenic fates and provide a rigorous framework for assessing in vivo glial reprogramming strategies.

AAV↗

Two-particle Aharonov-Bohm effect and entanglement in the electronic Hanbury Brown-Twiss setup.

We analyze a Hanbury Brown-Twiss geometry in which particles are injected from two independent sources into a mesoscopic conductor in the quantum Hall regime. All partial waves end in different reservoirs without generating any single-particle interference; in particular, there is no single-particle Aharonov-Bohm effect. However, exchange effects lead to two-particle Aharonov-Bohm oscillations in the zero-frequency current cross correlations. We demonstrate that this is related to two-particle orbital entanglement, detected via violation of a Bell inequality. The transport is along edge states and only adiabatic quantum point contacts and normal reservoirs are employed.

Journal Article↗

Postnucleation orbital implants.

A follow-up study of a group of 40 patients after the enucleation completed by the intra-orbital acrylic spherical magnetic implant (Roper-Hall style) is presented. The mean postoperative observation time is 3.3 years. Late complications were present in two patients: the extrusion of the implant in one of them was probably caused by his poor state due to chronic dialysis, while the traumatic luxation of the implant in the other was surgically repaired. Postoperative results in all other patients were satisfactory with the adequate orbital volume substitution and the ability of the prosthesis to twist and turn in any axis without fitting problems.

Adolescent↗

Magnetic relaxation dispersion studies of biomolecular solutions.

Although the MRD method has a long record in biomolecular systems, it has undergone a renaissance in the past few years as methodological developments have provided access to new types of information. In particular, MRD studies of quadrupolar nuclei such as 17O and 23Na have yielded valuable insights about the interactions of proteins and oligonucleotides with their solvent environment. The biomolecular MRD literature is still dominated by hydration studies, but the method has also been used to study the interaction of organic cosolvents and inorganic counterions with biomolecules. The MRD method can potentially make important contributions to the understanding of the mechanisms whereby protein conformational stability is affected by nonaqueous solvent components, such as denaturants, stabilizers, and helix promoters. Residence times of water molecules and other low molecular weight species in association with biomolecules can be determined by MRD. Such residence times are of general interest for understanding the kinetics of biomolecule-ligand interactions and, when exchange is gated by the biomolecule, can be used to characterize large-scale conformational fluctuations on nanosecond-millisecond time scales. By monitoring the integrity and specific internal hydration sites as well as the global solvent exposure, the MRD method can also shed light on the structure and dynamics of biomolecules in fluctuating nonnative states. Because it does not rely on high resolution, the MRD method is also applicable to very large biomolecules and complexes and has even been used to investigate protein crystals, gels, and biological tissues. In fact, dynamic studies of solids and liquid crystals were among the earliest applications of the MRD method. In many of its diverse applications, the MRD method provides unique information, complementing that available from high-resolution NMR.

Data Interpretation, Statistical↗

Surveillance of pneumococcal disease in Australian states and territories.

Information on pneumococcal disease, including immunisation programs, and optimum future surveillance in each Australian State and Territory were discussed at the Pneumococcal Disease in Australia Workshop on 26-27 March 1999. Workshop participants further expanded on the surveillance aspects of the Workshop in this report. Most participants favoured notification by laboratories of pneumococcal isolates from sterile sites, to provide baseline surveillance data before immunisation programs are fully implemented. It was also thought that trends in antimicrobial resistance should be notified.

Australia↗

Stripe formation in the fractional quantum Hall regime.

An understanding of the physics of the half-filled lowest Landau level has been achieved in terms of a Fermi sea of composite fermions, but the nature of the state at other even-denominator fractions has remained unclear. We investigate theoretically Landau level fillings of the form nu = (2n + 1)/(4n + 4), which correspond to composite-fermion fillings nu* = n + 1/2. By considering various plausible candidate states with complete spin polarization, we rule out the composite-fermion Fermi sea and paired composite-fermion state at these filling factors, and predict that the system phase separates into stripes of n and n + 1 filled Landau levels of composite fermions.

Journal Article↗

DNA sequence context and multiplex hybridization reactions: melting studies of heteromorphic duplex DNA complexes.

Heteromorphic hybrid duplex DNA complexes are duplex states, other than perfectly matched duplexes, that can form when single strands comprising several different perfectly matched duplexes are simultaneously present in solution. Such cross-hybridization "side reactions" are of particular nuisance in multiplex reaction schemes, where many strands are designed to hybridize in parallel fashion with only their corresponding perfect complement strand. Relative to the perfect match duplexes, the sequence dependent features of these heteromorphic duplex states and their thermodynamic stability are an important consideration for multiplex hybridization reaction design. We have measured absorbance versus temperature melting curves and performed differential scanning calorimetry measurements on various mixtures of eight different 24 base single strands. When perfect complementary pairs of strands are mixed in single reactions, four perfectly matched duplexes form. When mixtures of strands that are not perfectly matched are prepared and analyzed, melting transitions for cross-hybridization are observed along with significant hyperchromicity changes. This is indicative of a melting hybrid, heteromorphic duplex states formed from two nonperfectly matched strands. In addition, when both the perfectly matched and noncomplementary strands are mixed together (in multiplex hybridization reactions) at molar ratios of 1:1, 3:1, and 1:3, evidence of perfect duplex and heteromorphic duplex complexes is found in all cases. A new analytical tool for considering heterogeneous, duplex complexes in multiplex hybridization mixtures is presented and employed to interpret the acquired melting data.

Base Pair Mismatch↗

Youth and digital media: a policy research agenda.

At a time when researchers are still sorting out the complex relationship between adolescents and the mass media, the entire nature of the media system is undergoing dramatic change. The explosive growth of the Internet is ushering in a new digital media culture. Youth are embracing the new technologies much more rapidly than adults. In addition, because of their increased spending power, youth have become a valuable target market for advertisers. These trends have spurred the proliferation of Web sites and other forms of new-media content specifically designed for teens and children. The burgeoning digital marketplace has spawned a new generation of market research companies, and market research on children and youth is outpacing academic research on youth and the newer media. The emergence of this new media culture holds both promise and peril for youth. Whether the positive or negative vision of the digital future prevails will be determined, in large part, by decisions being made now and in the next few years in the halls of government and in corporate boardrooms. Research has contributed to the resolutions of several recent legislative and policy decisions in areas including television violence and the V-chip, children's educational television programming, and privacy and marketing to children on the Web. Future research needs to be designed with the public policy agenda in mind. The academic community has much to contribute to the debates over new developments in the digital age.

Adolescent↗

Coexistence of composite bosons and composite fermions in nu = 1/2 + 1/2 quantum Hall bilayers.

In bilayer quantum Hall systems at filling fractions near nu=1/2+1/2, as the spacing d between the layers is continuously decreased, intralayer correlations must be replaced by interlayer correlations, and the composite fermion (CF) Fermi seas at large d must eventually be replaced by a composite boson (CB) condensate or "111 state" at small d. We propose a scenario where CBs and CFs coexist in two interpenetrating fluids in the transition. Trial wave functions describing these mixed CB-CF states compare very favorably with exact diagonalization results. A Chern-Simons transport theory is constructed that is compatible with experiment.

Journal Article↗

Decreased mitochondrial creatine kinase activity in dystrophic chicken breast muscle alters creatine-linked respiratory coupling.

Dystrophic chicken breast muscle mitochondria contain significantly less mitochondrial creatine kinase than normal breast muscle mitochondria. Breast muscle mitochondria from normal 16- to 40-day-old chickens contain approximately 80 units of mitochondrial creatine kinase per unit of succinate:INT (p-iodonitrotetrazolium violet) reductase, a mitochondrial marker, while dystrophic chicken breast muscle mitochondria contain 36-44 units. Normal chicken heart muscle mitochondria contain about 10% of the mitochondrial creatine kinase per unit of succinate:INT reductase as normal breast muscle mitochondria. The levels in heart muscle mitochondria from dystrophic chickens are not affected significantly. Evidence is presented which shows that the reduced level of mitochondrial creatine kinase in dystrophic breast muscle mitochondria is responsible for an altered creatine linked respiration. First, both normal and dystrophic breast muscle mitochondria respire with the same state 3 and state 4 respiration. Second, the post-ADP state 4 rate of respiration of normal breast muscle mitochondria in the presence of 20 mM creatine continues at the state 3 rate. However, the state 4 rate of dystrophic breast muscle mitochondria and mitochondria from other muscle types with a low level of mitochondrial creatine kinase, such as heart muscle and 5-day-old chicken breast muscle, is slower than the state 3 rate. Third, dystrophic breast mitochondria synthesize ATP at the same rate as normal breast muscle mitochondria but rates of creatine phosphate synthesis in 20-50 mM Pi are reduced significantly. Finally, increasing concentrations of Pi displace mitochondrial creatine kinase from mitoplasts of normal and dystrophic breast muscle mitochondria with the same apparent KD, indicating that the outer surface of the inner mitochondrial membrane and the mitochondrial creatine kinase from dystrophic muscle are not altered.

Adenosine Triphosphate↗

Increased expression of glycolysis-associated genes in oncogene-transformed and growth-accelerated states.

An accelerated rate of glucose transport and catabolism is a common characteristic of cellular transformation. We have previously found elevated expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in human pancreatic and colonic adenocarcinomas (Schek et al.: Cancer Res 48:6354-6359, 1988). To investigate further the expression of this enzyme in the process of tumorigenesis, we examined GAPDH expression in a panel of oncogene-transformed fibroblasts. Significant elevations of GAPDH mRNA and glucose transporter protein mRNA levels were observed in ras- and mos-transformed NIH 3T3 cells, whereas little or no change was found in c-src-, v-src-, c-myc-, E1A-, v-fos-, and PKC-gamma-transfected cells. Furthermore, the level of GAPDH mRNA correlated with the transformed state in a series of ras-transformed and revertant cell lines. Immunoblot analysis confirmed that GAPDH polypeptide was significantly elevated in the cell lines with elevated mRNA levels. Cell cycle analysis data suggested that the effect on GAPDH expression correlated with oncogene expression rather than cell growth fraction. These results suggest that altered GAPDH gene expression occurs during some growth deregulated states, and this, along with increased glucose transporter (and possibly other glycolytic enzyme) expression, is likely to contribute to the increased metabolic capacity of cells in these states.

Animals↗

Effects of vaginally administered high estradiol doses on hormonal pharmacokinetics and hemostasis in postmenopausal women.

OBJECTIVE: To study the effect of high doses of estradiol released from vaginal rings on the pharmacokinetics of hormones, and the long-term effect on hormones and hemostasis in postmenopausal women. DESIGN: A pilot, nonrandomized study. SETTING: Healthy volunteers in an academic research environment. PATIENTS: Postmenopausal women. Eight women were treated with 17 beta-estradiol from three vaginal rings, releasing 7.5 micro g per ring for a total of 22.5 micro g over 24 hours. The rings were changed every morning for 14 days. MAIN OUTCOME MEASURE(S): Hemostatic changes were recorded. RESULT(S): Estradiol was rapidly absorbed, and statistically significant increases in the levels were found after 15 minutes; C(max) was obtained after 1 hour and a steady state after 24 hours. No statistically significant changes were found in the levels of coagulation factors V, von Willebrand factor, and activated factor VII; nor were any changes observed for activated protein C resistance, coagulation inhibitors protein C, protein S, or plasminogen activator inhibitor-1. No indication of increased thrombin formation was demonstrated by analyses of prothrombin fragment 1+2, fibrin D-dimer, and soluble fibrin. CONCLUSION(S)No statistically significant changes in hemostasis were observed from the vaginal administration of estradiol using a dose equivalent to transdermal administration with a release rate of 100 micro g per 24 hours.

Administration, Intravaginal↗

Evolution of quasiparticle charge in the fractional quantum hall regime

The charge of quasiparticles in a fractional quantum Hall (FQH) liquid, tunneling through a partly reflecting constriction with transmission t, was determined via shot noise measurements. In the nu = 1/3 FQH state, a charge smoothly evolving from e(*) = e/3 for t(1/3) congruent with 1 to e(*) = e for t(1/3)<<1 was determined, agreeing with chiral Luttinger liquid theory. In the nu = 2/5 FQH state the quasiparticle charge evolves smoothly from e(*) = e/5 at t(2/5) congruent with 1 to a maximum charge less than e(*) = e/3 at t(2/5)<<1. Thus it appears that quasiparticles with an approximate charge e/5 pass a barrier they see as almost opaque.

Journal Article↗

Pathobiology of dynorphins in trauma and disease.

Dynorphins, endogenous opioid neuropeptides derived from the prodynorphin gene, are involved in a variety of normative physiologic functions including antinociception and neuroendocrine signaling, and may be protective to neurons and oligodendroglia via their opioid receptor-mediated effects. However, under experimental or pathophysiological conditions in which dynorphin levels are substantially elevated, these peptides are excitotoxic largely through actions at glutamate receptors. Because the excitotoxic actions of dynorphins require supraphysiological concentrations or prolonged tissue exposure, there has likely been little evolutionary pressure to ameliorate the maladaptive, non-opioid receptor mediated consequences of dynorphins. Thus, dynorphins can have protective and/or proapoptotic actions in neurons and glia, and the net effect may depend upon the distribution of receptors in a particular region and the amount of dynorphin released. Increased prodynorphin gene expression is observed in several disease states and disruptions in dynorphin processing can accompany pathophysiological situations. Aberrant processing may contribute to the net negative effects of dysregulated dynorphin production by tilting the balance towards dynorphin derivatives that are toxic to neurons and/or oligodendroglia. Evidence outlined in this review suggests that a variety of CNS pathologies alter dynorphin biogenesis. Such alterations are likely maladaptive and contribute to secondary injury and the pathogenesis of disease.

Animals↗

Beta-actin mRNA localization is regulated by signal transduction mechanisms.

Beta-actin mRNA is localized in the leading lamellae of chicken embryo fibroblasts (CEFs) (Lawrence, J., and R. Singer. 1986. Cell. 45:407-415), close to where actin polymerization in the lamellipodia drives cellular motility. During serum starvation beta-actin mRNA becomes diffuse and non-localized. Addition of FCS induces a rapid (within 2-5 min) redistribution of beta-actin mRNA into the leading lamellae. A similar redistribution was seen with PDGF, a fibroblast chemotactic factor. PDGF-induced beta-actin mRNA redistribution was inhibited by the tyrosine kinase inhibitor herbimycin, indicating that this process requires intact tyrosine kinase activity, similar to actin filament polymerization and chemotaxis. Lysophosphatidic acid, which has been shown to rapidly induce actin stress fiber formation (Ridley, A., and A. Hall. 1992. Cell. 790:389-399), also increases peripheral beta-actin mRNA localization within minutes. This suggests that actin polymerization and mRNA localization may be regulated by similar signaling pathways. Additionally, activators or inhibitors of kinase A or C can also delocalize steady-state beta-actin mRNA in cells grown in serum, and can inhibit the serum induction of peripherally localized beta-actin mRNA in serum-starved CEFs. These data show that physiologically relevant extracellular factors operating through a signal transduction pathway can regulate spatial sites of actin protein synthesis, which may in turn affect cellular polarity and motility.

Actins↗