bluuebunny/crossref_metadata_2025 huggingface dataset:
A Cost-Based Strategy to Design Multiple Shell and Tube Heat Exchangers
Authors: Moita Raquel D. , Fernandes Cristina , Matos Henrique A. , Nunes Clemente P.
Published: March 2004

Process Integration has been applied in several industrial processes mainly using standard shell and tube heat exchangers (1-1 or 1-2). The flow arrangement in 1-2 multiple shell and tube heat exchangers involves part counter-current flow and part co-current flow. This fact is accounted for in the design by introducing a FT correction factor into the 1-1 heat exchanger design equation. To avoid some steep regions in the feasible space of heat exchangers design some authors introduce other parameters like XP or G. Until now it was not possible to have an overall map to give some guidelines of how to choose between the several XP approaches established in the literature. This paper summarizes the current existing criteria in a general design algorithm DeAl12 to show a path for the calculations of the main design variables of the heat exchanger. Also a new strategy design algorithm StratDeAl12 is introduced in this paper to allow the best choice between the existing XP approaches based on the heat exchanger cost minimisation. Several examples illustrate the advantage of using the developed algorithm and the deviations obtained in the heat exchanger cost if a wrong approach was chosen.
Heat Transfer Mechanisms During Flow Boiling in Microchannels
Authors: Kandlikar Satish G.
Published: March 2004

The forces due to surface tension and momentum change during evaporation, in conjunction with the forces due to viscous shear and inertia, govern the two-phase flow patterns and the heat transfer characteristics during flow boiling in microchannels. These forces are analyzed in this paper, and two new nondimensional groups, K1 and K2, relevant to flow boiling phenomenon are derived. These groups are able to represent some of the key flow boiling characteristics, including the CHF. In addition, a mechanistic description of the flow boiling phenomenon is presented. The small hydraulic dimensions of microchannel flow passages present a large frictional pressure drop in single-phase and two-phase flows. The small hydraulic diameter also leads to low Reynolds numbers, in the range 100–1000, or even lower for smaller diameter channels. Such low Reynolds numbers are rarely employed during flow boiling in conventional channels. In these low Reynolds number flows, nucleate boiling systematically emerges as the dominant mode of heat transfer. The high degree of wall superheat required to initiate nucleation in microchannels leads to rapid evaporation and flow instabilities, often resulting in flow reversal in multiple parallel channel configuration. Aided by strong evaporation rates, the bubbles nucleating on the wall grow rapidly and fill the entire channel. The contact line between the bubble base and the channel wall surface now becomes the entire perimeter at both ends of the vapor slug. Evaporation occurs at the moving contact line of the expanding vapor slug as well as over the channel wall covered with a thin evaporating film surrounding the vapor core. The usual nucleate boiling heat transfer mechanisms, including liquid film evaporation and transient heat conduction in the liquid adjacent to the contact line region, play an important role. The liquid film under the large vapor slug evaporates completely at downstream locations thus presenting a dryout condition periodically with the passage of each large vapor slug. The experimental data and high speed visual observations confirm some of the key features presented in this paper.
Laminar Forced Convection Heat Transfer in the Combined Entry Region of Non-Circular Ducts
Authors: Muzychka Y. S. , Yovanovich M. M.
Published: March 2004

A new model for predicting Nusselt numbers in the combined entrance region of non-circular ducts and channels is developed. This model predicts both local and average Nusselt numbers and is valid for both isothermal and isoflux boundary conditions. The model is developed using the asymptotic results for convection from a flat plate, thermally developing flows in non-circular ducts, and fully developed flow in non-circular ducts. Through the use of a novel characteristic length scale, the square root of cross-sectional area, the effect of duct shape on Nusselt number is minimized. Comparisons are made with several existing models for the circular tube and parallel plate channel and with numerical data for several non-circular ducts. Agreement between the proposed model and numerical data is within ±15percent or better for most duct shapes.
A Numerical Study of Natural Convection in Partially Open Enclosures With a Conducting Side-Wall
Authors: Desrayaud G. , Lauriat G.
Published: March 2004

A numerical study of natural convection generated by a cold vertical wall of an enclosure with two openings on the opposite wall of finite thickness is presented. The enclosure is connected to an infinite reservoir filled with hot air. A two-dimensional laminar flow is assumed both within the enclosure and along the side of the bounding wall immersed into the reservoir. The effects of the size of the openings, spacing between the vertical walls and thermal resistance of the bounding wall are investigated. Numerical results are discussed for aspect ratios of the enclosure and Rayleigh numbers relevant to practical applications.
Modeling and Numerical Prediction of Flow Boiling in a Thin Geometry
Authors: Kumar Ranganathan , Maneri Charles C. , Strayer T. Darton
Published: March 2004

An analysis capability to examine the two-phase bubbly flow in high pressure boiling systems has been developed. The models have been adapted from the literature for a narrow high aspect ratio geometry using the measurements obtained in a companion paper. Three-dimensional computational results have been compared with cross-section averaged and line-averaged void fractions measured with a gamma densitometer, and local void fraction measured with a hot-film anemometer. These comparisons have been made over a wide range of flow inlet conditions, wall heating and system pressure. Comparisons are found to be good when the flow is bubbly, but at high void fractions, where the flow is churn-turbulent or annular, the two-field modeling approach does not perform adequately. This result emphasizes the need for multiple field modeling.
Effect of Ethoxylation and Molecular Weight of Cationic Surfactants on Nucleate Boiling in Aqueous Solutions
Authors: Zhang Juntao , Manglik Raj M.
Published: March 2004

Saturated, nucleate pool boiling on a horizontal, cylindrical heater and the associated bubble dynamics in aqueous solutions of cationic surfactants of different molecular weight and ethoxylation or ethylene oxide (EO) content, are experimentally investigated. Boiling curves qw″∝ΔTsat for different concentrations and photographic records of the salient features of the ebullient behavior are presented, along with a characterization of interfacial properties (surface tension and contact angle). The surfactant additive significantly alters the nucleate boiling in water and enhances the heat transfer. The enhancement increases with concentration, with an optimum obtained in solutions at or near the critical micelle concentration (c.m.c.) of the surfactant. The photographic and visual observations indicate a markedly different boiling behavior than that of water, as well as between pre- and post-c.m.c. solutions. A lower molecular weight surfactant tends to reduce surface tension faster, and show better enhancement performance than its higher molecular weight counterpart. With EO groups in its molecular chain the surfactant solution becomes more hydrophilic, and the higher wettability tends to suppress nucleation, thereby weakening the boiling process. Also, enhancement in pre-micellar solutions is shown to depend on the dynamic surface tension, and the number of EO groups in and molecular weight of the surfactant.
Fluctuating Temperature Measurements on a Heated Cylinder Placed in a Cylinder Near-Wake
Authors: Wang Z. J. , Zhou Y. , Wang X. W. , Jin W.
Published: March 2004

The local time-averaged temperature θs¯ and its fluctuating component θs on the surface of a heated circular cylinder immersed in a cylinder near-wake were measured using a fiber-optic Bragg grating (FBG) sensor. Three cylinder center-to-center spacing, i.e., L/d=5.20, 2.50, and 1.18, were investigated. In order to validate the FBG sensor measurement, a thermocouple and a single hot-wire were employed to measure θs¯ on the heated cylinder and streamwise fluctuating velocity u in the near-wake of the downstream cylinder, respectively. The FBG sensor measurement of θs¯ is in good agreement with that simultaneously obtained by the thermocouple. The measured θs is closely correlated to the hot-wire measurement; the θs-spectrum exhibits a pronounced peak at the vortex shedding frequency, as identified in Eu, for each L/d. The results suggest that the FBG sensor can be used to measure reliably both time-averaged and fluctuating temperatures. The heat transfer characteristics of the heated cylinder are examined for different L/d and further compared with the case of an isolated cylinder.
From Mechanisms and Robotics to Protein Conformation and Drug Design
Authors: Kazerounian Kazem
Published: March 2004

The systematic study of kinematics can be traced to the writings of the ancient Greeks, Egyptians, Romans and Persians as far back as 500 B.C. For many centuries kinematics (along with geometry) was regarded as one of the basic sciences that explained observed physical phenomena and was used to engineer machines. Though it may seem unlikely, kinematics (in particular, robot kinematics) can significantly contribute to our understanding of biological systems and their functions at the microscopic level and to the engineering of new diagnostic tools, treatments, and drugs for a variety of diseases. Given the vast body of knowledge in theoretical, applied, and analytical kinematics and robotics, the conspicuous absence of the kinematics community from current molecular science research relating to the prediction of protein folding, protein docking, protein engineering, and drug design seems puzzling. In this paper, we will discuss the potential contributions of kinematics to some current challenges in biotechnology.
Square-wave excitation of a linear oscillator
Authors: Butikov Eugene I.
Published: March 2004

The forced oscillations of a torsion spring pendulum excited by an external square-wave external torque are discussed. Two different ways of determining the steady-state response of the oscillator are described and compared. The behavior of this familiar mechanical system can help a student better understand why and how a LCR-circuit transfers a square-wave voltage from input to output with a distortion of its shape.
Precision measurements of a simple chaotic circuit
Authors: Kiers Ken , Schmidt Dory , Sprott J. C.
Published: March 2004

We describe a simple nonlinear electrical circuit that can be used to study chaotic phenomena. The circuit employs simple electronic elements such as diodes, resistors, and operational amplifiers, and is easy to construct. A novel feature of the circuit is its use of an almost ideal nonlinear element, which is straightforward to model theoretically and leads to excellent agreement between experiment and theory. For example, comparisons of bifurcation points and power spectra give agreement to within 1%. The circuit yields a broad range of behavior and is well suited for qualitative demonstrations and as a serious research tool.
Oscillator damped by a constant-magnitude friction force
Authors: Marchewka Avi , Abbott David S. , Beichner Robert J.
Published: March 2004

Although a simple spring/mass system damped by a friction force of constant magnitude shares many of the characteristics of the simple and damped harmonic oscillators, its solution is not presented in most texts. Closed form solutions for the turning and stopping points can be found using an energy-based approach. A dynamical approach leads to a closed form solution for the position of the mass as a function of time. The main result is that the amplitude of the oscillator damped by a constant magnitude friction force decreases by a constant amount each swing and the motion dies out after a finite time. We present these two solutions and suggest ways that the system can be used in the classroom.
From conservation of energy to the principle of least action: A story line
Authors: Hanc Jozef , Taylor Edwin F.
Published: March 2004

We outline a story line that introduces Newtonian mechanics by employing conservation of energy to predict the motion of a particle in a one-dimensional potential. We show that incorporating constraints and constants of the motion into the energy expression allows us to analyze more complicated systems. A heuristic transition embeds kinetic and potential energy into the still more powerful principle of least action.
A simple model for stochastic coherence and stochastic resonance
Authors: Hilborn Robert C.
Published: March 2004

I describe a simple iterated map that displays two important noise-induced effects for nonlinear systems: stochastic coherence and stochastic resonance. The model requires only modest computational capabilities and some knowledge of nonlinear dynamics and illustrates the constructive role of noise in nonlinear systems.
Getting the most action out of least action: A proposal
Authors: Moore Thomas A.
Published: March 2004

Lagrangian methods lie at the foundation of contemporary theoretical physics. Several recent articles have explored the possibility of making the principle of least action and Lagrangian methods a part of the first-year physics curriculum. I examine some of this proposal’s implications for subsequent courses in the undergraduate physics major, and focus on the influence that this proposal might have on the selection of topics and the opportunities this proposal presents for teaching these courses in a more contemporary way. Many of these ideas are relevant even if students first learn Lagrangian methods in a sophomore mechanics course.
Modern mechanics
Authors: Chabay Ruth W. , Sherwood Bruce A.
Published: March 2004

We consider the goals of the introductory course in classical mechanics taken by physics majors and argue both that these goals are not well met in actual courses and that the goals themselves should be rethought. We propose alternative goals and describe an introductory “modern mechanics” course that addresses these alternative goals. Included in the description are several genres of homework problems that are nearly absent from traditional mechanics courses at both the introductory and intermediate levels. The intermediate mechanics course could be restructured to exploit a broader foundation laid by the introductory course.
Synchronous analog I/O for acquisition of chaotic data in periodically driven systems
Authors: DeSerio Robert
Published: March 2004

A new data acquisition technique has been designed for measuring chaotic attractors in periodically driven systems. Analog output hardware generates a continuous periodic waveform used in the drive while analog input hardware digitizes one or more chaotic waveforms characterizing the system phase space variables. With both processes synchronized to a common clock, the construction of multiple Poincaré sections is reduced to the simple process of redimensioning the chaotic waveforms and taking slices in one of the array indices. The implementation described here is applied to an electronic Duffing oscillator running at 1.6 kHz and provides quality Poincaré sections in near real time.
Weakly nonlinear oscillations: A perturbative approach
Authors: Kahn Peter B. , Zarmi Yair
Published: March 2004

The perturbative analysis of a one-dimensional harmonic oscillator subject to a small nonlinear perturbation is developed within the framework of two popular methods: normal forms and multiple time scales. The systems analyzed are the Duffing oscillator, an energy conserving oscillatory system, the cubically damped oscillator, a system that exhibits damped oscillations, and the Van der Pol oscillator, which represents limit-cycle systems. Special emphasis is given to the exploitation of the freedom inherent in the calculation of the higher-order terms in the expansion and to the comparison of the application of the two methods to the three systems.
Microbial Polysaccharides Template Assembly of Nanocrystal Fibers
Authors: Chan Clara S. , De Stasio Gelsomina , Welch Susan A. , Girasole Marco , Frazer Bradley H. , Nesterova Maria V. , Fakra Sirine , Banfield Jillian F.
Published: March 2004

Biological systems can produce extraordinary inorganic structures and morphologies. The mechanisms of synthesis are poorly understood but are of great interest for engineering novel materials. We use spectromicroscopy to show that microbially generated submicrometer-diameter iron oxyhydroxide (FeOOH) filaments contain polysaccharides, providing an explanation for the formation of akaganeite pseudo–single crystals with aspect ratios of ∼1000:1. We infer that the cells extrude the polysaccharide strands to localize FeOOH precipitation in proximity to the cell membrane to harness the proton gradient for energy generation. Characterization of organic compounds with high spatial resolution, correlated with mineralogical information, should improve our understanding of biomineralization mechanisms.
De Novo Crypt Formation and Juvenile Polyposis on BMP Inhibition in Mouse Intestine
Authors: Haramis Anna-Pavlina G. , Begthel Harry , van den Born Maaike , van Es Johan , Jonkheer Suzanne , Offerhaus G. Johan A. , Clevers Hans
Published: March 2004

Little is known about the signaling mechanisms that determine the highly regular patterning of the intestinal epithelium into crypts and villi. With the use of mouse models, we show that bone morphogenetic protein (BMP)–4 expression occurs exclusively in the intravillus mesenchyme. Villus epithelial cells respond to the BMP signal. Inhibition of BMP signaling by transgenic expression of noggin results in the formation of numerous ectopic crypt units perpendicular to the crypt-villus axis. These changes phenocopy the intestinal histopathology of patients with the cancer predisposition syndrome juvenile polyposis (JP), including the frequent occurrence of intraepithelial neoplasia. Many JP cases are known to harbor mutations in BMP pathway genes. These data indicate that intestinal BMP signaling represses de novo crypt formation and polyp growth.
Elastomeric Transistor Stamps: Reversible Probing of Charge Transport in Organic Crystals
Authors: Sundar Vikram C. , Zaumseil Jana , Podzorov Vitaly , Menard Etienne , Willett Robert L. , Someya Takao , Gershenson Michael E. , Rogers John A.
Published: March 2004

We introduce a method to fabricate high-performance field-effect transistors on the surface of freestanding organic single crystals. The transistors are constructed by laminating a monolithic elastomeric transistor stamp against the surface of a crystal. This method, which eliminates exposure of the fragile organic surface to the hazards of conventional processing, enables fabrication of rubrene transistors with charge carrier mobilities as high as ∼15 cm
2
/V·s and subthreshold slopes as low as 2nF·V/decade·cm
2
. Multiple relamination of the transistor stamp against the same crystal does not affect the transistor characteristics; we exploit this reversibility to reveal anisotropic charge transport at the basal plane of rubrene.
The Wheat
VRN2
Gene Is a Flowering Repressor Down-Regulated by Vernalization
Authors: Yan Liuling , Loukoianov Artem , Blechl Ann , Tranquilli Gabriela , Ramakrishna Wusirika , SanMiguel Phillip , Bennetzen Jeffrey L. , Echenique Viviana , Dubcovsky Jorge
Published: March 2004

Plants with a winter growth habit flower earlier when exposed for several weeks to cold temperatures, a process called vernalization. We report here the positional cloning of the wheat vernalization gene
VRN2,
a dominant repressor of flowering that is down-regulated by vernalization. Loss of function of
VRN2
, whether by natural mutations or deletions, resulted in spring lines, which do not require vernalization to flower. Reduction of the RNA level of
VRN2
by RNA interference accelerated the flowering time of transgenic winter-wheat plants by more than a month.
Dissipationless Anomalous Hall Current in the Ferromagnetic Spinel CuCr
2
Se

4-
x

Br

x
Authors: Lee Wei-Li , Watauchi Satoshi , Miller V. L. , Cava R. J. , Ong N. P.
Published: March 2004

In a ferromagnet, an applied electric field E invariably produces an anomalous Hall current J
H
that flows perpendicular to the plane defined by E and M (the magnetization). For decades, the question of whether J
H
is dissipationless (independent of the scattering rate) has been debated without experimental resolution. In the ferromagnetic spinel CuCr
2
Se
4–

x

Br

x

, the resistivity ρ (at low temperature) may be increased by several decades by varying
x
(Br) without degrading M. We show that J
H
/E (normalized per carrier, at 5 kelvin) remains unchanged throughout. In addition to confirming the dissipationless nature of J
H
, our finding has implications for the generation and study of spin-Hall currents in bulk samples.
Direct Spinning of Carbon Nanotube Fibers from Chemical Vapor Deposition Synthesis
Authors: Li Ya-Li , Kinloch Ian A. , Windle Alan H.
Published: March 2004

Many routes have been developed for the synthesis of carbon nanotubes, but their assembly into continuous fibers has been achieved only through postprocessing methods. We spun fibers and ribbons of carbon nanotubes directly from the chemical vapor deposition (CVD) synthesis zone of a furnace using a liquid source of carbon and an iron nanocatalyst. This process was realized through the appropriate choice of reactants, control of the reaction conditions, and continuous withdrawal of the product with a rotating spindle used in various geometries. This direct spinning from a CVD reaction zone is extendable to other types of fiber and to the spin coating of rotating objects in general.
Controlled Atomic Doping of a Single C
60
Molecule
Authors: Yamachika R. , Grobis M. , Wachowiak A. , Crommie M. F.
Published: March 2004

We report a method for controllably attaching an arbitrary number of charge dopant atoms directly to a single, isolated molecule. Charge-donating K atoms adsorbed on a silver surface were reversibly attached to a C
60
molecule by moving it over K atoms with a scanning tunneling microscope tip. Spectroscopic measurements reveal that each attached K atom donates a constant amount of charge (∼0.6 electron charge) to the C
60
host, thereby enabling its molecular electronic structure to be precisely and reversibly tuned.
Health Care in the Service of Science?
Authors: Reinhardt Uwe E.
Published: March 2004

What Price Better Health?
Hazards of the Research Imperative. by Daniel Callahan. University of California Press, Berkeley, and The Milbank Memorial Fund, New York, 2003. 341 pp. $29.95, £19.95. ISBN 0-520-22771-9.

Callahan's exploration of the history, conduct, and goals of biomedical research in the United States questions the claims that more science improves our lives and that aging is a preventable disease.
Transmembrane Segments of Syntaxin Line the Fusion Pore of Ca
2
+-Triggered Exocytosis
Authors: Han Xue , Wang Chih-Tien , Bai Jihong , Chapman Edwin R. , Jackson Meyer B.
Published: March 2004

The fusion pore of regulated exocytosis is a channel that connects and spans the vesicle and plasma membranes. The molecular composition of this important intermediate structure of exocytosis is unknown. Here, we found that mutations of some residues within the transmembrane segment of syntaxin (Syx), a plasma membrane protein essential for exocytosis, altered neurotransmitter flux through fusion pores and altered pore conductance. The residues that influenced fusion-pore flux lay along one face of an α-helical model. Thus, the fusion pore is formed at least in part by a circular arrangement of 5 to 8 Syx transmembrane segments in the plasma membrane.
Azido-Containing Diketo Acid Derivatives Inhibit Human Immunodeficiency Virus Type 1 Integrase In Vivo and Influence the Frequency of Deletions at Two-Long-Terminal-Repeat-Circle Junctions
Authors: Svarovskaia Evguenia S. , Barr Rebekah , Zhang Xuechun , Pais Godwin C. G. , Marchand Christophe , Pommier Yves , Burke Terrence R. , Pathak Vinay K.
Published: March 2004

We previously found that azido-containing β-diketo acid derivatives (DKAs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase (IN) (X. Zhang et al., Bioorg. Med. Chem. Lett., 13:1215-1219, 2003). To characterize the intracellular mechanisms of action of DKAs, we analyzed the antiviral activities of two potent azido-containing DKAs with either a monosubstitution or a disubstitution of azido groups, using single- and multiple-replication-cycle assays. Both azido-containing DKAs significantly inhibited HIV-1 infection in 293T, CEM-SS, and H9 cells (50% inhibitory concentration = 2 to 13 μM) and exhibited low cytotoxicity (50% cytotoxic concentration = 60 to 600 μM). Inhibition of HIV-1 IN in vivo was demonstrated by the observation that previously described L-708,906 resistance mutations in HIV-1 IN (T66I and T66I/S153Y) also conferred resistance to the azido-group-containing DKAs. In vitro assays and in vivo analysis indicated that the DKAs did not significantly inhibit the 3′ processing and selectively inhibited the strand transfer reaction. In addition, quantitative PCR indicated that two-long-terminal-repeat (2-LTR) circles were elevated in the presence of the azido-containing DKAs, confirming that HIV-1 IN was the intracellular target of viral inhibition. To gain insight into the mechanism by which the DKAs increased 2-LTR-circle formation of 3′-processed viral DNAs, we performed extensive DNA sequencing analysis of 2-LTR-circle junctions. The results indicated that the frequency of deletions at the circle junctions was elevated from 19% for the untreated controls to 32 to 41% in the presence of monosubstituted (but not disubstituted) DKAs. These results indicate that the structure of the DKAs can influence the extent of degradation of viral DNA ends by host nucleases and the frequency of deletions at the 2-LTR-circle junctions. Thus, sequencing analysis of 2-LTR-circle junctions can elucidate the intracellular mechanisms of action of HIV-1 IN inhibitors.
Cleavage of Eukaryotic Translation Initiation Factor 4GII within Foot-and-Mouth Disease Virus-Infected Cells: Identification of the L-Protease Cleavage Site In Vitro
Authors: Gradi Alessandra , Foeger Nicole , Strong Rebecca , Svitkin Yuri V. , Sonenberg Nahum , Skern Tim , Belsham Graham J.
Published: March 2004

Foot-and-mouth disease virus (FMDV) induces a very rapid inhibition of host cell protein synthesis within infected cells. This is accompanied by the cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI). The cleavage of the related protein eIF4GII has now been analyzed. Within FMDV-infected cells, cleavage of eIF4GI and eIF4GII occurs with similar kinetics. Cleavage of eIF4GII is induced in cells and in cell extracts by the FMDV leader protease (L
pro
) alone, generating cleavage products similar to those induced by enterovirus and rhinovirus 2A protease (2A
pro
). By the use of a fusion protein containing residues 445 to 744 of human eIF4GII, it was demonstrated that the FMDV L
pro
specifically cleaves this protein between residues G700 and S701, immediately adjacent to the site (V699/G700) cleaved by rhinovirus 2A
pro
in vitro. The G700/S701 cleavage site does not correspond, by amino acid sequence alignment, to that cleaved in eIF4GI by the FMDV L
pro
in vitro. Knowledge of the cleavage sites and the three-dimensional structures of the FMDV L
pro
and rhinovirus 2A
pro
enabled mutant forms of the eIF4GII sequence to be generated that are differentially resistant to either one of these proteases. These results confirmed the specificity of each protease and showed that the mutant forms of the fusion protein substrate retained their correct sensitivity to other proteases.
p16
Ink4a
Interferes with Abelson Virus Transformation by Enhancing Apoptosis
Authors: Sachs Zohar , Sharpless Norman E. , DePinho Ronald A. , Rosenberg Naomi
Published: March 2004

Pre-B-cell transformation by Abelson virus (Ab-MLV) is a multistep process in which primary transformants are stimulated to proliferate but subsequently undergo crisis, a period of erratic growth marked by high levels of apoptosis. Inactivation of the p53 tumor suppressor pathway is an important step in this process and can be accomplished by mutation of
p53
or down-modulation of p19
Arf
, a p53 regulatory protein. Consistent with these data, pre-B cells from either
p53
or
Ink4a/Arf
null mice bypass crisis. However, the
Ink4a/Arf
locus encodes both p19
Arf
and a second tumor suppressor, p16
Ink4a
, that blocks cell cycle progression by inhibiting Cdk4/6. To determine if p16
Ink4a
plays a role in Ab-MLV transformation, primary transformants derived from
Arf
−/−
and
p16

Ink4a
−/−

mice were compared. A fraction of those derived from
Arf
−/−
animals underwent crisis, and even though all
p16

Ink4a
−/−

primary transformants experienced crisis, these cells became established more readily than cells derived from +/+ mice. Analyses of
Ink4a/Arf
−/−
cells infected with a virus that expresses both v-Abl and p16
Ink4a
revealed that p16
Ink4a
expression does not alter cell cycle profiles but does increase the level of apoptosis in primary transformants. These results indicate that both products of the
Ink4a/Arf
locus influence Ab-MLV transformation and reveal that in addition to its well-recognized effects on the cell cycle, p16
Ink4a
can suppress transformation by inducing apoptosis.
Preextinction Viral RNA Can Interfere with Infectivity
Authors: González-López Claudia , Arias Armando , Pariente Nonia , Gómez-Mariano Gema , Domingo Esteban
Published: March 2004

When the error rate during the copying of genetic material exceeds a threshold value, the genetic information cannot be maintained. This concept is the basis of a new antiviral strategy termed lethal mutagenesis or virus entry into error catastrophe. Critical for its success is preventing survival of residual infectious virus or virus mutants that escape the transition into error catastrophe. Here we document that mutated, preextinction foot-and-mouth disease virus (FMDV) RNA can interfere with and delay viral production up to 30 h when cotransfected in BHK-21 cells with standard RNA. Interference depended on the physical integrity of preextinction RNA and was not observed with unrelated RNAs or with nonmutated, defective FMDV RNA. These results suggest that this type of interference requires large size, preextinction FMDV RNA and is mediated neither by small interfering RNAs nor by RNAs that can compete with infectious RNA for host cell factors. A model based on the aberrant expression of mutated RNA as it is expected to occur in the initial stages of the transition into error catastrophe is proposed. Interference mediated by preextinction RNA indicates an advantage of mutagenesis versus inhibition in preventing the survival of virus escape mutants during antiviral treatments.
The Poliovirus Replication Machinery Can Escape Inhibition by an Antiviral Drug That Targets a Host Cell Protein
Authors: Crotty Shane , Saleh Maria-Carla , Gitlin Leonid , Beske Oren , Andino Raul
Published: March 2004

Viral replication depends on specific interactions with host factors. For example, poliovirus RNA replication requires association with intracellular membranes. Brefeldin A (BFA), which induces a major rearrangement of the cellular secretory apparatus, is a potent inhibitor of poliovirus RNA replication. Most aspects governing the relationship between viral replication complex and the host membranes remain poorly defined. To explore these interactions, we used a genetic approach and isolated BFA-resistant poliovirus variants. Mutations within viral proteins 2C and 3A render poliovirus resistant to BFA. In the absence of BFA, viruses containing either or both of these mutations replicated similarly to wild type. In the presence of BFA, viruses carrying a single mutation in 2C or 3A exhibited an intermediate-growth phenotype, while the double mutant was fully resistant. The viral proteins 2C and 3A have critical roles in both RNA replication and vesicle formation. The identification of BFA resistant mutants may facilitate the identification of cellular membrane-associated proteins necessary for induction of vesicle formation and RNA replication. Importantly, our data underscore the dramatic plasticity of the host-virus interactions required for successful viral replication.
Nonnucleoside Inhibitor Binding Affects the Interactions of the Fingers Subdomain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with DNA
Authors: Peletskaya Elena N. , Kogon Alex A. , Tuske Steven , Arnold Edward , Hughes Stephen H.
Published: March 2004

Site-directed photoaffinity cross-linking experiments were performed by using human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with unique cysteine residues at several positions (i.e., positions 65, 67, 70, and 74) in the fingers subdomain of the p66 subunit. Since neither the introduction of the unique cysteine residues into the fingers nor the modification of the SH groups of these residues with photoaffinity cross-linking reagents caused a significant decrease in the enzymatic activities of RT, we were able to use this system to measure distances between specific positions in the fingers domain of RT and double-stranded DNA. HIV-1 RT is quite flexible. There are conformational changes associated with binding of the normal substrates and nonnucleoside RT inhibitors (NNRTIs). Cross-linking was used to monitor intramolecular movements associated with binding of an NNRTI either in the presence or in the absence of an incoming deoxynucleoside triphosphate (dNTP). Binding an incoming dNTP at the polymerase active site decreased the efficiency of cross-linking but caused only modest changes in the preferred positions of cross-linking. This finding suggests that the fingers of p66 are closer to an extended template in the “open” configuration of the enzyme with the fingers away from the active site than in the closed configuration with the fingers in direct contact with the incoming dNTP. NNRTI binding caused increased cross-linking in experiments with diazirine reagents (especially with a diazirine reagent with a longer linker) and a moderate shift in the preferred sites of interaction with the template. Cross-linking occurred closer to the polymerase active site for RTs modified at positions 70 and 74. The effects of NNRTI binding were more pronounced in the absence of a bound dNTP; pretreatment of HIV-1 RT with an NNRTI reduced the effect of dNTP binding. These observations can be explained if the binding of NNRTI causes a decrease in the flexibility in the fingers subdomain of RT-NNRTI complex and a decrease in the distance from the fingers to the template extension.
Coronavirus Neurovirulence Correlates with the Ability of the Virus To Induce Proinflammatory Cytokine Signals from Astrocytes and Microglia
Authors: Li Yun , Fu Li , Gonzales Donna M. , Lavi Ehud
Published: March 2004

The molecular and cellular basis of coronavirus neurovirulence is poorly understood. Since neurovirulence may be determined at the early stages of infection of the central nervous system (CNS), we hypothesize that it may depend on the ability of the virus to induce proinflammatory signals from brain cells for the recruitment of blood-derived inflammatory cells. To test this hypothesis, we studied the interaction between coronaviruses (mouse hepatitis virus) of different neurovirulences with primary cell cultures of brain immune cells (astrocytes and microglia) and mouse tissues. We found that the level of neurovirulence of the virus correlates with its differential ability to induce proinflammatory cytokines (interleukin 12 [IL-12] p40, tumor necrosis factor alpha, IL-6, IL-15, and IL-1β) in astrocytes and microglia and in mouse brains and spinal cords. These findings suggest that coronavirus neurovirulence may depend on a novel discriminatory ability of astrocytes and microglia to induce a proinflammatory response in the CNS.
Human Immunodeficiency Virus Type 1 Transductive Recombination Can Occur Frequently and in Proportion to Polyadenylation Signal Readthrough
Authors: An Wenfeng , Telesnitsky Alice
Published: March 2004

One model for retroviral transduction suggests that template switching between viral RNAs and polyadenylation readthrough sequences is responsible for the generation of acute transforming retroviruses. For this study, we examined reverse transcription products of human immunodeficiency virus (HIV)-based vectors designed to mimic postulated transduction intermediates. For maximization of the discontinuous mode of DNA synthesis proposed to generate transductants, sequences located between the vectors' two long terminal repeats (vector “body” sequences) and polyadenylation readthrough “tail” sequences were made highly homologous. Ten genetic markers were introduced to indicate which products had acquired tail sequences by a process we term transductive recombination. Marker segregation patterns for over 100 individual products were determined, and they revealed that more than half of the progeny proviruses were transductive recombinants. Although most crossovers occurred in regions of homology, about 5% were nonhomologous and some included insertions. Ratios of encapsidated readthrough and polyadenylated transcripts for vectors with wild-type and inactivated polyadenylation signals were compared, and transductive recombination frequencies were found to correlate with the readthrough transcript prevalence. In assays in which either vector body or tail could serve as a recombination donor, recombination between tail and body sequences was at least as frequent as body-body exchange. We propose that transductive recombination may contribute to natural HIV variation by providing a mechanism for the acquisition of nongenomic sequences.
Coupling of Human Immunodeficiency Virus Type 1 Fusion to Virion Maturation: a Novel Role of the gp41 Cytoplasmic Tail
Authors: Wyma Donald J. , Jiang Jiyang , Shi Jiong , Zhou Jing , Lineberger Janet E. , Miller Michael D. , Aiken Christopher
Published: March 2004

Retrovirus particles are not infectious until they undergo proteolytic maturation to form a functional core. Here we report a link between human immunodeficiency virus type 1 (HIV-1) core maturation and the ability of the virus to fuse with target cells. Using a recently developed reporter assay of HIV-1 virus-cell fusion, we show that immature HIV-1 particles are 5- to 10-fold less active for fusion with target cells than are mature virions. The fusion of mature and immature virions was rendered equivalent by truncating the gp41 cytoplasmic domain or by pseudotyping viruses with the glycoprotein of vesicular stomatitis virus. An analysis of a panel of mutants containing mutated cleavage sites indicated that HIV-1 fusion competence is activated by the cleavage of Gag at any site between the MA and NC segments and not as an indirect consequence of an altered core structure. These results suggest a mechanism by which binding of the gp41 cytoplasmic tail to Gag within immature HIV-1 particles inhibits Env conformational changes on the surface of the virion that are required for membrane fusion. This “inside-out” regulation of HIV-1 fusion could play an important role in the virus life cycle by preventing the entry of immature, noninfectious particles.
High-Resolution Phylogenetic Analysis of Hepatitis C Virus Adaptation and Its Relationship to Disease Progression
Authors: Sheridan Isabelle , Pybus Oliver G. , Holmes Edward C. , Klenerman Paul
Published: March 2004

Hepatitis C virus (HCV) persists in the majority of those infected despite host immune responses. Evidence has accrued that selectively fixed mutations in the envelope genes (E1 and E2) are associated with viral persistence, particularly those that occur within the first hypervariable region of E2 (HVR1). However, the individual amino acid residues under selection have not been identified, nor have their selection pressures been measured, despite the importance of this information for understanding disease pathogenesis and for vaccine design. We performed a high-resolution analysis of published gene sequence data from individuals undergoing acute HCV infection, employing two phylogenetic methods to determine site-specific selection pressures. Strikingly, we found a statistically significant association between the number of sites selected and disease outcome, with the fewest selected sites in fulminant HCV cases and the greatest number of selected sites in rapid progressors, reflecting the duration and intensity of the arms race between host and virus. Moreover, sites outside the HVR1 appear to play a major role in viral evolution and pathogenesis, although there was no association between viral persistence and specific mutations in E1 and E2. Our analysis therefore allows fine dissection of immune selection pressures, which may be more diverse than previously thought. Such analyses could play a similarly informative role in studies of other persistent virus infections, such as human immunodeficiency virus.
Spacers Increase the Accessibility of Peptide Ligands Linked to the Carboxyl Terminus of Adenovirus Minor Capsid Protein IX
Authors: Vellinga Jort , Rabelink Martijn J. W. E. , Cramer Steve J. , van den Wollenberg Diana J. M. , Van der Meulen Hans , Leppard Keith N. , Fallaux Frits J. , Hoeben Rob C.
Published: March 2004

The efficiency and specificity of gene transfer with human adenovirus (hAd)-derived gene transfer vectors would be improved if the native viral tropism could be modified. Here, we demonstrate that the minor capsid protein IX (pIX), which is present in 240 copies in the Ad capsid, can be exploited as an anchor for heterologous polypeptides. Protein IX-deleted hAd5 vectors were propagated in hAd5 helper cells expressing pIX variants, with heterologous carboxyl-terminal extensions of up to 113 amino acids in length. The extensions evaluated consist of alpha-helical spacers up to 75 Å in length and to which peptide ligands were fused. The pIX variants were efficiently incorporated into the capsids of Ad particles. On intact particles, the MYC-tagged-pIX molecules were readily accessible to anti-MYC antibodies, as demonstrated by electron microscopic analyses of immunogold-labeled virus particles. The labeling efficiency improved with increasing spacer length, suggesting that the spacers lift and expose the ligand at the capsid surface. Furthermore, we found that the addition of an integrin-binding RGD motif to the pIX markedly stimulated the transduction of coxsackievirus group B and hAd receptor-deficient endothelioma cells, demonstrating the utility of pIX modification in gene transfer. Our data demonstrate that the minor capsid protein IX can be used as an anchor for the addition of polypeptide ligands to Ad particles.
High-Throughput Screening of the Yeast Kinome: Identification of Human Serine/Threonine Protein Kinases That Phosphorylate the Hepatitis C Virus NS5A Protein
Authors: Coito Carlos , Diamond Deborah L. , Neddermann Petra , Korth Marcus J. , Katze Michael G.
Published: March 2004

The hepatitis C virus NS5A protein plays a critical role in virus replication, conferring interferon resistance to the virus through perturbation of multiple intracellular signaling pathways. Since NS5A is a phosphoprotein, it is of considerable interest to understand the role of phosphorylation in NS5A function. In this report, we investigated the phosphorylation of NS5A by taking advantage of 119 glutathione
S
-transferase-tagged protein kinases purified from
Saccharomyces cerevisiae
to perform a global screening of yeast kinases capable of phosphorylating NS5A in vitro. A database BLAST search was subsequently performed by using the sequences of the yeast kinases that phosphorylated NS5A in order to identify human kinases with the highest sequence homologies. Subsequent in vitro kinase assays and phosphopeptide mapping studies confirmed that several of the homologous human protein kinases were capable of phosphorylating NS5A. In vivo phosphopeptide mapping revealed phosphopeptides common to those generated in vitro by AKT, p70S6K, MEK1, and MKK6, suggesting that these kinases may phosphorylate NS5A in mammalian cells. Significantly, rapamycin, an inhibitor commonly used to investigate the mTOR/p70S6K pathway, reduced the in vivo phosphorylation of specific NS5A phosphopeptides, strongly suggesting that p70S6 kinase and potentially related members of this group phosphorylate NS5A inside the cell. Curiously, certain of these kinases also play a major role in mRNA translation and antiapoptotic pathways, some of which are already known to be regulated by NS5A. The findings presented here demonstrate the use of high-throughput screening of the yeast kinome to facilitate the major task of identifying human NS5A protein kinases for further characterization of phosphorylation events in vivo. Our results suggest that this novel approach may be generally applicable to the screening of other protein biochemical activities by mechanistic class.
Purification and Crystallization Reveal Two Types of Interactions of the Fusion Protein Homotrimer of Semliki Forest Virus
Authors: Gibbons Don L. , Reilly Brigid , Ahn Anna , Vaney Marie-Christine , Vigouroux Armelle , Rey Felix A. , Kielian Margaret
Published: March 2004

The fusion proteins of the alphaviruses and flaviviruses have a similar native structure and convert to a highly stable homotrimer conformation during the fusion of the viral and target membranes. The properties of the alpha- and flavivirus fusion proteins distinguish them from the class I viral fusion proteins, such as influenza virus hemagglutinin, and establish them as the first members of the class II fusion proteins. Understanding how this new class carries out membrane fusion will require analysis of the structural basis for both the interaction of the protein subunits within the homotrimer and their interaction with the viral and target membranes. To this end we report a purification method for the E1 ectodomain homotrimer from the alphavirus Semliki Forest virus. The purified protein is trimeric, detergent soluble, retains the characteristic stability of the starting homotrimer, and is free of lipid and other contaminants. In contrast to the postfusion structures that have been determined for the class I proteins, the E1 homotrimer contains the fusion peptide region responsible for interaction with target membranes. This E1 trimer preparation is an excellent candidate for structural studies of the class II viral fusion proteins, and we report conditions that generate three-dimensional crystals suitable for analysis by X-ray diffraction. Determination of the structure will provide our first high-resolution views of both the low-pH-induced trimeric conformation and the target membrane-interacting region of the alphavirus fusion protein.