bluuebunny/crossref_metadata_2025 huggingface dataset:
SoMART: a web server for plant miRNA, tasiRNA and target gene analysis
Authors: Li Feng , Orban Ryan , Baker Barbara
Published: January 2012

Plant microRNAs (miRNAs) and trans‐acting small interfering RNAs (tasiRNAs) play important roles in a variety of biological processes. Bioinformatics prediction and small RNA (sRNA) cloning are the most important approaches for identification of miRNAs and tasiRNAs and their targets. However, these approaches are not readily accessible to every researcher. Here we present SoMART, a web server for miRNA/tasiRNA analysis resources and tools, which is designed for researchers who are interested in identifying miRNAs or tasiRNAs that potentially regulate genes of interest. The server includes four sets of tools: ‘Slicer detector’ for detecting sRNAs targeting input genes, ‘dRNA mapper’ for detecting degradome (d)RNA products derived from input genes, ‘PreMIR detector’ for identifying miRNA precursors (MIRs) or tasiRNA precursor (TASs) of input sRNAs, and ‘sRNA mapper’ for mapping sRNAs onto input genes. We also developed a dRNA‐seq protocol to achieve longer dRNA reads for better characterization of miRNA precursors by dRNA mapper. To validate the server function and robustness, we installed sRNA, dRNA and collected genomic DNA or transcriptome databases from Arabidopsis and solanaceous plants, and characterized miR172‐mediated regulation of the APETALA2 gene in potato (Solanum tuberosum) and demonstrated conservation of MIR390‐triggered TAS3 in tomato (Solanum lycopersicum). More importantly, we predicted the existence of MIR482‐triggered TAS5 in tomato. We further tested and confirmed the efficiency and accuracy of the server by analyses of 21 validated miRNA targets and 115 miRNA precursors in Arabidopsis thaliana. SoMART is available at http://somart.ist.berkeley.edu.
Ecological radiation with limited morphological diversification in salamanders
Authors: BLANKERS T. , ADAMS D. C. , WIENS J. J.
Published: January 2012

A major goal of evolutionary biology is to explain morphological diversity among species. Many studies suggest that much morphological variation is explained by adaptation to different microhabitats. Here, we test whether morphology and microhabitat use are related in plethodontid salamanders, which contain the majority of salamander species, and have radiated into a striking diversity of microhabitats. We obtained microhabitat data for 189 species that also had both morphometric and phylogenetic data. We then tested for associations between morphology and microhabitat categories using phylogenetic comparative methods. Associations between morphology and ecology in plethodontids are largely confined to a single clade within one subfamily (Bolitoglossinae), whereas variation in morphology across other plethodontids is unrelated to microhabitat categories. These results demonstrate that ecological radiation and morphological evolution can be largely decoupled in a major clade. The results also offer a striking contrast to lizards, which typically show close relationships between morphology and microhabitat.
Morphological differentiation correlates with ecological but not with genetic divergence in aGehyragecko
Authors: SISTROM M. , EDWARDS D.L. , DONNELLAN S. , HUTCHINSON M.
Published: January 2012

Body size affects life history, the ecological niche of an organism and its interactions with other organisms. Resultantly, marked differences in body size between related organisms are often an indication of a species boundary. This is particularly evident in theGehyra variegataspecies complex of geckos, which displays differential body sizes between genetically divergent species, but high levels of intraspecific morphological conservatism. We report on aGehyrapopulation that displays extraordinary body size differentiation in comparison with otherG. variegataspecies. We used morphological and environmental data to show this population is phenotypically and ecologically distinct from its parapatric congenerGehyra lazelliand that morphology and ecology are significantly correlated. Contrastingly, mtDNA analysis indicates paraphyly between the two groups, and allele frequencies at six microsatellite loci show no population structure concordant with morpho‐/ecotype. These results suggest either ecological speciation or environmentally induced phenotypic polymorphism, in an otherwise morphologically conservative group.
In children using intensive insulin therapy, a 20‐g variation in carbohydrate amount significantly impacts on postprandial glycaemia
Authors: Smart C. E. , King B. R. , McElduff P. , Collins C. E.
Published: January 2012

Diabet. Med. 29, e21–e24 (2012)

Aim  To determine if an insulin dose calculated for a meal containing 60 g carbohydrate maintains postprandial glycaemic control for meals containing 40, 50, 70 or 80 g carbohydrate.

Methods  Thirty‐four young people (age range 8.5–17.7 years) using intensive insulin therapy consumed five test breakfasts with equivalent fat, protein and fibre contents but differing carbohydrate quantities (40, 50, 60, 70 and 80 g of carbohydrate). The preprandial insulin dose was the same for each meal, based on the subject’s usual insulin:carbohydrate ratio for 60 g carbohydrate. Continuous glucose monitoring was used to monitor postprandial glucose over 180 min.

Results  The 40‐g carbohydrate meal resulted in significantly more hypoglycaemia than the other meals (P = 0.003). There was a one in three chance of hypoglycaemia between 120 and 180 min if an insulin dose for 60 g carbohydrate was given for 40 g carbohydrate. The glucose levels of subjects on the 80‐g meal were significantly higher than the 60‐ and 70‐g carbohydrate meals at all time points between 150 and 180 min (P < 0.01). Subjects consuming the 80‐g meal were more likely to have significant hyperglycaemia (blood glucose levels ≥ 12 mmol/l) compared with the other meals (P < 0.001).

Conclusions  In patients using intensive insulin therapy, an individually calculated insulin dose for 60 g carbohydrate results in postprandial hypoglycaemia or hyperglycaemia for meals containing 40 and 80 g carbohydrate. To calculate mealtime insulin in order to maintain postprandial control, carbohydrate estimations should be within 10 g of the actual meal carbohydrate.
Rattling cisticola song features and variability across sub‐Saharan Africa
Authors: Benedict L. , Bowie R. C. K.
Published: January 2012

Quantifying bird song variation can be an important tool for ensuring accurate species identification and can provide a significant basis for understanding the evolutionary processes that shape phenotypic diversity. This study describes variation in the songs of rattling cisticolas Cisticola chiniana across sub‐Saharan Africa. For many cisticola species, learned songs are the most obvious phenotypic indicators of species affiliation and may also function to indicate individual quality. We examined 957 songs recorded from 61 individuals and archived in sound libraries. To assess the diversity of syllable and song types, we examined patterns of syllable use. We also measured vocalization frequency and time parameters and assessed how they vary through space. Results indicated that rattling cisticola songs are highly variable, but also have features that are species‐specific. Examined songs had a relatively fixed structure containing one of three characteristic introductory note types, followed by an end phrase. Two of the introductory note types were sung across the species’ range (some 4500 km), whereas the third was only recorded in south‐western Africa. End phrases generated most of the diversity in songs and appeared to have an unlimited number of forms. End‐phrase characteristics showed a strong geographic variation, but did not vary with elevation. Song features varied individually and geographically in ways that are consistent with evolution due to multiple selective pressures, including stabilizing selection for species recognition on the introductory notes and diversifying selection on the end phrases. This pattern of lability in some song features coupled with stability in others may be a common feature of cisticola songs as it has also been found in Cisticola erythrops, a congener with a similarly broad range.
The Unique Features of Traumatic Brain Injury in Children. Review of the Characteristics of the Pediatric Skull and Brain, Mechanisms of Trauma, Patterns of Injury, Complications and Their Imaging Findings—Part 1
Authors: Pinto Pedro S. , Poretti Andrea , Meoded Avner , Tekes Aylin , Huisman Thierry A.G.M.
Published: January 2012

Traumatic head/brain injury (TBI) is a leading cause of death and life‐long disability in children. The biomechanical properties of the child's brain and skull, the size of the child, the age‐specific activity pattern, and higher degree of brain plasticity result in a unique distribution, degree, and quality of TBI compared to adult TBI. A detailed knowledge about the various types of primary and secondary pediatric head injuries is essential to better identify and understand pediatric TBI. The goals of this review article are (1) to discuss the unique epidemiology, mechanisms, and characteristics of TBI in children, and (2) to review the anatomical and functional imaging techniques that can be used to study common and rare pediatric traumatic brain injuries and their complications. J Neuroimaging 2012;22:e1–e17.
Resveratrol‐Mediated Downregulation of Rictor Attenuates Autophagic Process and Suppresses UV‐Induced Skin Carcinogenesis†
Authors: Back Jung H. , Zhu Yucui , Calabro Alyssa , Queenan Craig , Kim Audrey S. , Arbesman Joshua , Kim Arianna L.
Published: January 2012

Macroautophagy is a cellular response to various environmental stresses that ensures lysosomal degradation of long‐lived and damaged proteins and cellular organelles. It occurs through the formation of an autophagosome, which then fuses with a lysosome to form an autolysosome. Depending on the cellular context, autophagy may promote cancer cell survival or it may serve as a mechanism of tumor suppression. Herein, we show that resveratrol, a natural phytoalexin, induces premature senescence in human A431 SCC cells, and that resveratrol‐induced premature senescence is associated with a blockade of autolysosome formation, as assessed by the absence of colocalization of LC3 and Lamp‐2, markers for autophagosomes and lysosomes, respectively. Further, we show that resveratrol downregulates the level of Rictor, a component of mTORC2, leading to decreased RhoA‐GTPase and altered actin cytoskeleton organization. Exogenous overexpression of Rictor restores RhoA‐GTPase activity and actin cytoskeleton network, and decreases resveratrol‐induced senescence‐associated β‐gal activity, indicating a direct role of Rictor in senescence induction. Rictor is overexpressed in UV‐induced murine SCCs, whereas its expression is diminished by oral administration of resveratrol. These data indicate that resveratrol attenuates autophagic process via Rictor, and suggest that downregulation of Rictor may be a mechanism of tumor suppression associated with premature senescence.
Creating Community Collaboration to Improve the Care of Patients With High Blood Pressure: Lessons From Rochester, New York
Authors: Bisognano John D. , Speranza Paul S. , Becker Lawrence M. , Norwood Wade S. , Bradley Al , Nazar Michael D. , Beckman Howard B.
Published: January 2012

J Clin Hypertens (Greenwich). 2012;14:178–183. ©2012 Wiley Periodicals, Inc.

Over the past two years, the business community of Monroe County, which includes Rochester, New York, has been engaging in a collaborative to improve outcomes for people with high blood pressure. As the employers examined the costs of care in the community, they recognized two important factors. First, the costs of care for the uninsured, the underinsured, and the Medicare population influence the business community's cost of care. Second, trying to redesign care just for their employees alone was not effective. This project is unique in that the stimulus and funding for community‐wide action comes from the business community. They saw beyond the often unsuccessful short‐term cost reduction programs and joined with a community‐focused organization, the Finger Lakes Health Systems Agency, to construct a multi‐year, multi‐faceted intervention designed to encourage practice redesign and an invigorated community commitment to partnership and accountability. This report describes the process to date and hopefully will stimulate conversations about mechanisms to encourage similar collaboration within other communities.
Universal primers for fluorescent labelling of PCR fragments—an efficient and cost‐effective approach to genotyping by fluorescence
Authors: BLACKET M. J. , ROBIN C. , GOOD R. T. , LEE S. F. , MILLER A. D.
Published: January 2012

Directly labelling locus‐specific primers for microsatellite analysis is expensive and a common limitation to small‐budget molecular ecology projects. More cost‐effective end‐labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus‐specific primers with 5′ universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co‐amplifying large numbers of size overlapping loci and without requiring locus‐specific PCR conditions to be modified. In this study, we report a suite of four high‐performance universal primers that can be employed in a three primer PCR approach for efficient and cost‐effective fluorescent end‐labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co‐amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.
Genetic Polymorphism of NAT2 Metabolizing Enzymes on Phenytoin Pharmacokinetics in Indian Epileptic Patients Developing Toxicity
Authors: Murali M. , Manjari T. , Madhuri B. , Raghavan S. , Jain D.C. , Vivekanandhan S.
Published: January 2012

SUMMARY  Objective: To investigate the effects of NAT2 metabolizing enzymes on the pharmacokinetics of antiepileptic drug phenytoin in the epileptic patients showing toxicity. Methods: Fifty epileptic individuals who had developed toxicity to phenytoin and 50 control epileptic subjects who had not developed toxicity to phenytoin were genotyped for NAT2 (NAT2*5A, NAT2*5C, NAT2*7, NAT2*6) polymorphisms by polymerase chain reaction–restriction fragment length polymorphisms (PCR–RFLP method). Phenytoin plasma levels were analyzed by reversed phase HPLC method and pharmacokinetic parameters such as area under the concentration curve (AUC), maximum concentration (Cmax), time to Cmax (tmax) and half‐life (t1/2) were estimated by noncompartmental analysis using PK Solutions® software. Results: The NAT2 polymorphism was seen to be in Hardy–Weinberg equilibrium and showed significant genotypic as well as allelic association with phenytoin toxicity for NAT2*5A (481C>T) and NAT2*5C (803A>G). Pharmacokinetic parameters for phenytoin in toxicity group of poor metabolizers showed a longer elimination half‐life of a drug (t1/2= 35.3 h) and less clearance rate (CL = 468 mL/h) compared to intermediate metabolizers (t1/2= 33.2 h, CL = 674 mL/h) and extensive metabolizer (t1/2= 20.7 h, CL = 977 mL/h) in NAT2*5A polymorphism. Conclusion: Our findings suggest that the NAT2*5A genetic polymorphisms plays a significant role in the steady‐state concentrations of phenytoin and thereby have impact on toxicity in epileptic patients.
NichePy: modular tools for estimating the similarity of ecological niche and species distribution models
Authors: Bentlage Bastian , Shcheglovitova Mariya
Published: January 2012

1. Ecological niche and species distribution models (ENMs and SDMs) are widely used in macroecological studies to investigate the potential geographic distributions of invasive species, the effects of climate change on past, present and future species distributions as well as the evolution of ecological niches. For many of these studies, estimating the similarity of different ENMs and SDMs is critical to evaluate the robustness of models.

2. We implement previously described randomization tests for evaluating the similarity of ENMs and SDMs in a package of three programs/scripts using the Python Programming Language. Our implementation is modular and flexible. It can be used with a variety of existing ENM and SDM algorithms and can be integrated into high‐throughput analyses using a command‐line shell (e.g. the Bash shell that is found on many Unix‐like operating systems).

3. We demonstrate the application of NichePy using distributional data of two Anolis species from Hispaniola. An analysis integrating NichePy and openModeller, including a simple and straight‐forward approach to ensemble modelling, is outlined.
Changes in correlation between spontaneous activity of dorsal horn neurones lead to differential recruitment of inhibitory pathways in the cat spinal cord
Authors: Chávez D. , Rodríguez E. , Jiménez I. , Rudomin P.
Published: January 2012

Key points 

We have examined the functional organization of the neuronal ensembles involved in the generation of spontaneous cord dorsum potentials in the lumbo‐sacral spinal cord of the anaesthetized cat.

These potentials appear synchronously along several spinal segments and are generated by a longitudinally distributed network of bilaterally interconnected sets of dorsal horn neurones.

Low levels of synchronization of spontaneous neuronal activity within this network appear associated with activation of spinal pathways mediating glycinergic non‐reciprocal postsynaptic inhibition of motoneurones.

During states of spontaneous increased synchronization, or after the acute section of cutaneous nerves, there is a preferential activation of the GABAergic pathways producing primary afferent depolarization and presynaptic inhibition of muscle and cutaneous afferents.

It is suggested that modulation of the temporal synchronization of spontaneous activity of dorsal horn neurones might provide means for selection of alternatively operating inhibitory spinal pathways during different sensory and motor behaviours.
Finite Element Aortic Injury Reconstruction of Near Side Lateral Impacts Using Real World Crash Data
Authors: Belwadi Aditya , Siegel John H. , Singh Aadarsh , Smith Joyce A. , Yang King H. , King Albert I.
Published: January 2012

Traumatic rupture of the aorta (TRA) remains the second most common cause of death associated with motor vehicle crashes, only less prevalent than brain injury. On average, nearly 8000 people die annually in the United States due to blunt injury to the aorta. It is observed that over 80% of occupants who suffer an aortic injury die at the scene due to exsanguination into the chest cavity. In the current study, eight near side lateral impacts, in which TRA occurred, were reconstructed using a combination of real world crash data reported in the Crash Injury Research and Engineering Network (CIREN) database, finite element (FE) models of vehicles, and the Wayne State Human Body Model - II (WSHBM). For the eight CIREN cases reconstructed, the high strain regions in the aorta closely matched with the autopsy data provided. The peak average maximum principal strains in all of the eight CIREN cases were localized in the isthmus region of the aorta, distal to the left subclavian artery, and averaged at 22 ± 6.2% while the average maximum pressure in the aorta was found to be 117 ± 14.7 kPa.
Using In Vivo Cine and 3D Multi-Contrast MRI to Determine Human Atherosclerotic Carotid Artery Material Properties and Circumferential Shrinkage Rate and Their Impact on Stress/Strain Predictions
Authors: Liu Haofei , Canton Gador , Yuan Chun , Yang Chun , Billiar Kristen , Teng Zhongzhao , Hoffman Allen H. , Tang Dalin
Published: January 2012

In vivo magnetic resonance image (MRI)-based computational models have been introduced to calculate atherosclerotic plaque stress and strain conditions for possible rupture predictions. However, patient-specific vessel material properties are lacking in those models, which affects the accuracy of their stress/strain predictions. A noninvasive approach of combining in vivo Cine MRI, multicontrast 3D MRI, and computational modeling was introduced to quantify patient-specific carotid artery material properties and the circumferential shrinkage rate between vessel in vivo and zero-pressure geometries. In vivo Cine and 3D multicontrast MRI carotid plaque data were acquired from 12 patients after informed consent. For each patient, one nearly-circular slice and an iterative procedure were used to quantify parameter values in the modified Mooney-Rivlin model for the vessel and the vessel circumferential shrinkage rate. A sample artery slice with and without a lipid core and three material parameter sets representing stiff, median, and soft materials from our patient data were used to demonstrate the effect of material stiffness and circumferential shrinkage process on stress/strain predictions. Parameter values of the Mooney-Rivlin models for the 12 patients were quantified. The effective Young’s modulus (YM, unit: kPa) values varied from 137 (soft), 431 (median), to 1435 (stiff), and corresponding circumferential shrinkages were 32%, 12.6%, and 6%, respectively. Using the sample slice without the lipid core, the maximum plaque stress values (unit: kPa) from the soft and median materials were 153.3 and 96.2, which are 67.7% and 5% higher than that (91.4) from the stiff material, while the maximum plaque strain values from the soft and median materials were 0.71 and 0.293, which are about 700% and 230% higher than that (0.089) from the stiff material, respectively. Without circumferential shrinkages, the maximum plaque stress values (unit: kPa) from the soft, median, and stiff models were inflated to 330.7, 159.2, and 103.6, which were 116%, 65%, and 13% higher than those from models with proper shrinkage. The effective Young’s modulus from the 12 human carotid arteries studied varied from 137 kPa to 1435 kPa. The vessel circumferential shrinkage to the zero-pressure condition varied from 6% to 32%. The inclusion of proper shrinkage in models based on in vivo geometry is necessary to avoid over-estimating the stresses and strains by up 100%. Material stiffness had a greater impact on strain (up to 700%) than on stress (up to 70%) predictions. Accurate patient-specific material properties and circumferential shrinkage could considerably improve the accuracy of in vivo MRI-based computational stress/strain predictions.
Experimental Evidence of Impingement Induced Strains at the Interface and the Periphery of an Embedded Acetabular Cup Implant
Authors: Arndt Christoph , Voigt Christian , Steinke Hanno , Salis-Soglio Georg v. , Scholz Roger
Published: January 2012

After total hip arthroplasty, impingement of implant components may occur during every-day patient activities causing increased shear stresses at the acetabular implant–bone interface. In the literature, impingement related lever-out moments were noted for a number of acetabular components. But there is little information about pelvic load transfer. The aim of the current study was to measure the three-dimensional strain distribution at the macrostructured hemispherical interface and in the periphery of a standard acetabular press-fit cup in an experimental implant-bone substitute model. An experimental setup was developed to simulate impingement loading via a lever arm representing the femoral component and the lower limb. In one experimental setup 12 strain gauges were embedded at predefined positions in the periphery of the acetabular cup implant inside a tray, using polyurethane composite resin as a bone substitute material. By incremental rotation of the implant tray in steps of 10 and 30 deg, respectively, the strains were measured at evenly distributed positions. With the described method 288 genuine strain values were measured in the periphery of an embedded acetabular cup implant in one experimental setup. In two additional setups the strains were evaluated at different distances from the implant interface. Both in radial and meridional interface directions strain magnitudes reach their peak near the rim of the cup below the impingement site. Values of equatorial strains vary near zero and reach their peaks near the rim of the cup on either side and in some distance from the impingement site. Interestingly, the maximum of averaged radial strains does not occur, as expected, close to the interface but at an interface offset of 5.6 mm. With the described experimental setup it is now possible to measure and display the three-dimensional strain distribution in the interface and the periphery of an embedded acetabular cup implant. The current study provides the first experimental proof of the high local stresses gradients in the direct vicinity of the impingement site. The results of the current study help for a better understanding of the impingement mechanism and its impact on acetabular cup stability.
Investigation of Saccadic Eye Movement Effects on the Fluid Dynamic in the Anterior Chamber
Authors: Abouali Omid , Modareszadeh Amirreza , Ghaffarieh Alireza , Tu Jiyuan
Published: January 2012

The aqueous humor (AH) flow in the anterior chamber (AC) due to saccadic movements is investigated in this research. The continuity, Navier-Stokes and energy equations in 3D and unsteady forms are solved numerically and the saccadic motion was modeled by the dynamic mesh technique. Firstly, the numerical model was validated for the saccadic movement of a spherical cavity with analytic solutions and experimental data where excellent agreement was observed. Then, two types of periodic and realistic saccadic motions of the AC are simulated, whereby the flow field is computed for various saccade amplitudes and the results are reported for different times. The results show that the acting shear stress on the corneal endothelial cells from AH due to saccadic movements is much higher than that due to normal AH flow by buoyancy induced due to temperature gradient. This shear stress is higher on the central region of the cornea. The results also depict that eye saccade imposes a 3D complicated flow field in the AC consist of various vortex structures. Finally, the enchantment of heat transfer in the AC by AH mixing as a result of saccadic motion is investigated.
A Pseudokinase Debut at the Mycobacterial Cell Wall
Authors: Warner Digby F. , Mizrahi Valerie
Published: January 2012

A serine-threonine kinase targets a pseudokinase to control the synthesis of the cell wall of the bacterium that causes tuberculosis.
Taking the Myc
Authors: Kiberstis Paula A.
Published: January 2012

An RNA interference screen identifies a “druggable” enzyme whose inhibition halts tumor cell growth.
Preserving a Pump with Insulin
Authors: Adler Elizabeth M.
Published: January 2012

Insulin switches pancreatic acinar cells to a primarily glycolytic metabolism under conditions of oxidative stress, allowing them to maintain ATP production.
Activity of a Novel Combined Antiretroviral Therapy of Gemcitabine and Decitabine in a Mouse Model for HIV-1
Authors: Clouser Christine L. , Holtz Colleen M. , Mullett Mary , Crankshaw Daune L. , Briggs Jacquie E. , O'Sullivan M. Gerard , Patterson Steven E. , Mansky Louis M.
Published: January 2012

The emergence of drug resistance threatens to limit the use of current anti-HIV-1 drugs and highlights the need to expand the number of treatment options available for HIV-1-infected individuals. Our previous studies demonstrated that two clinically approved drugs, decitabine and gemcitabine, potently inhibited HIV-1 replication in cell culture through a mechanism that is distinct from the mechanisms for the drugs currently used to treat HIV-1 infection. We further demonstrated that gemcitabine inhibited replication of a related retrovirus, murine leukemia virus (MuLV),
in vivo
using the MuLV-based LP-BM5/murine AIDS (MAIDS) mouse model at doses that were not toxic. Since decitabine and gemcitabine inhibited MuLV and HIV-1 replication with similar potency in cell culture, the current study examined the efficacy and toxicity of the drug combination using the MAIDS model. The data demonstrate that the drug combination inhibited disease progression, as detected by histopathology, viral loads, and spleen weights, at doses lower than those that would be required if the drugs were used individually. The combination of decitabine and gemcitabine exerted antiviral activity at doses that were not toxic. These findings indicate that the combination of decitabine and gemcitabine shows potent antiretroviral activity at nontoxic doses and should be further investigated for clinical relevance.
A dominant negative erythropoietin (EPO) receptor inhibits EPO-dependent growth and blocks F-gp55-dependent transformation.
Authors: Barber D L , DeMartino J C , Showers M O , D'Andrea A D
Published: January 2012

The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated to signal cell growth by binding either EPO or F-gp55, the Friend spleen focus-forming virus glycoprotein. Activation by F-gp55 results in constitutive EPO-R signalling and the first stage of Friend virus-induced erythroleukemia. We have generated a truncated form of the EPO-R polypeptide [EPO-R(T)] which lacks the critical cytoplasmic signal-transducing domain of the EPO-R required for EPO- or F-gp55-induced cell growth. EPO-R(T) specifically inhibited the EPO-dependent growth of EPO-R-expressing Ba/F3 cells without changing the interleukin-3-dependent growth of these cells. In addition, Ba/F3 cells that coexpressed wild-type EPO-R and EPO-R(T) were resistant to transformation by F-gp55 despite efficient expression of the F-gp55 transforming oncoprotein in infected cells. EPO-R(T) inhibited the EPO-dependent tyrosine phosphorylation of wild-type EPO-R, the tyrosine kinase (JAK2), and the SH2 adaptor protein (Shc). In conclusion, the EPO-R(T) polypeptide is a dominant negative polypeptide which specifically interferes with the early stages of EPO-R-mediated signal transduction and which prevents Friend virus transformation of erythroblasts.
A constitutively activated erythropoietin receptor stimulates proliferation and contributes to transformation of multipotent, committed nonerythroid and erythroid progenitor cells.
Authors: Longmore G D , Pharr P N , Lodish H F
Published: January 2012

If the env gene of spleen focus-forming virus (SFFV) is replaced by a cDNA encoding a constitutively active form of the erythropoietin receptor, EPO-R(R129C), the resultant recombinant virus, SFFVcEPO-R, induces transient thrombocytosis and erythrocytosis in infected mice. Clonogenic progenitor cell assays of cells from the bone marrow and spleens of these infected mice suggest that EPO-R(R129C) can stimulate proliferation of committed megakaryocytic and erythroid progenitors as well as nonerythroid multipotent progenitors. From the spleens of SFFVcEPO-R-infected mice, eight multiphenotypic immortal cell lines were isolated and characterized. These included primitive erythroid, lymphoid, and monocytic cells. Some expressed proteins characteristic of more than one lineage. All cell lines resulting from SFFVcEPO-R infection contained a mutant form of the p53 gene. However, in contrast to infection by SFFV, activation of PU.1 gene expression, by retroviral integration, was not observed. One cell line had integrated a provirus upstream of the fli-1 gene, in a location typically seen in erythroleukemic cells generated by Friend murine leukemia virus infection. This event led to increased expression of fli-1 in this cell line. Thus, infection by SFFVcEPO-R can induce proliferation and lead to transformation of nonerythroid as well as very immature erythroid progenitor cells. The sites of proviral integration in clonal cell lines are distinct from those in SFFV-derived lines.
Subcellular locations of MOD5 proteins: mapping of sequences sufficient for targeting to mitochondria and demonstration that mitochondrial and nuclear isoforms commingle in the cytosol.
Authors: Boguta M , Hunter L A , Shen W C , Gillman E C , Martin N C , Hopper A K
Published: January 2012

MOD5, a gene responsible for the modification of A37 to isopentenyl A37 of both cytosolic and mitochondrial tRNAs, encodes two isozymes. Initiation of translation at the first AUG of the MOD5 open reading frame generates delta 2-isopentenyl pyrophosphate:tRNA isopentanyl transferase I (IPPT-I), which is located predominantly, but not exclusively, in the mitochondria. Initiation of translation at a second AUG generates IPPT-II, which modifies cytoplasmic tRNA. IPPT-II is unable to target to mitochondria. The N-terminal sequence present in IPPT-I and absent in IPPT-II is therefore necessary for mitochondrial targeting. In these studies, we fused MOD5 sequences encoding N-terminal regions to genes encoding passenger proteins, pseudomature COXIV and dihydrofolate reductase, and studied the ability of these chimeric proteins to be imported into mitochondria both in vivo and in vitro. We found that the sequences necessary for mitochondrial import, amino acids 1 to 11, are not sufficient for efficient mitochondrial targeting and that at least some of the amino acids shared by IPPT-I and IPPT-II comprise part of the mitochondrial targeting information. We used indirect immunofluorescence and cell fractionation to locate the MOD5 isozymes in yeast. IPPT-I was found in two subcellular compartments: mitochondria and the cytosol. We also found that IPPT-II had two subcellular locations: nuclei and the cytosol. The nuclear location of this protein is surprising because the A37-->isopentenyl A37 modification had been predicted to occur in the cytoplasm. MOD5 is one of the first genes reported to encode isozymes found in three subcellular compartments.
A nuclear encoded tRNA of Trypanosoma brucei is imported into mitochondria.
Authors: Schneider A , Martin J , Agabian N
Published: January 2012

The mitochondrial genome of trypanosomes, unlike that of most other eukaryotes, does not appear to encode any tRNAs. Therefore, mitochondrial tRNAs must be either imported into the organelle or created through a novel mitochondrial process, such as RNA editing. Trypanosomal tRNA(Tyr), whose gene contains an 11-nucleotide intron, is present in both the cytosol and the mitochondrion and is encoded by a single-copy nuclear gene. By site-directed mutagenesis, point mutations were introduced into this tRNA gene, and the mutated gene was reintroduced into the trypanosomal nuclear genome by DNA transfection. Expression of the mutant tRNA led to the accumulation of unspliced tRNA(Tyr) (A. Schneider, K. P. McNally, and N. Agabian, J. Biol. Chem. 268:21868-21874, 1993). Cell fractionation revealed that a significant portion of the unspliced mutant tRNA(Tyr) was recovered in the mitochondrial fraction and was resistant to micrococcal nuclease treatment in the intact organelle. Expression of the nuclear integrated, mutated tRNA gene and recovery of its gene product in the mitochondrial fraction directly demonstrated import. In vitro experiments showed that the unspliced mutant tRNA(Tyr), in contrast to the spliced wild-type form, was no longer a substrate for the cognate aminoacyl synthetase. The presence of uncharged tRNA in the mitochondria demonstrated that aminoacylation was not coupled to import.
A segment of mRNA encoding the leader peptide of the CPA1 gene confers repression by arginine on a heterologous yeast gene transcript.
Authors: Delbecq P , Werner M , Feller A , Filipkowski R K , Messenguy F , Piérard A
Published: January 2012

The expression of the yeast gene CPA1, which encodes the small subunit of the arginine pathway carbamoylphosphate synthetase, is repressed by arginine at a translational level. CPA1 mRNA contains a 250-nucleotide-long leader which includes a 25-codon upstream open reading frame (uORF). Oligonucleotide site-directed mutagenesis of this uORF as well as sequencing of constitutive cis-dominant mutations has suggested that the leader peptide product of the CPA1 uORF is an essential negative element for repression of the CPA1 gene by arginine. In this work, a series of deletions affecting the regions 5' and 3' to the uORF in the leader sequence was constructed. The arginine-dependent repression of CPA1 was little affected in these constructions, indicating that these regions are not essential for the regulatory response. This conclusion was further supported by the finding that inserting the mRNA segment encoding the leader peptide sequence of CPA1 in the leader sequence of another gene, namely, GCN4, places this gene under arginine repression. Similarly, the behavior of fusions of the leader sequence of CPA1 with those of ARG4 or GAL10 confirmed that the regions of this leader located upstream and downstream from the uORF are dispensable for the regulation by arginine. Finally, a set of substitution mutations which modify the uORF nucleotide sequence while leaving unchanged the corresponding amino acid sequence was constructed. The mutations did not affect the repression of CPA1 by arginine. The data presented in this paper consequently agree with the conclusion that the leader peptide itself is the main element required for the translational repression of CPA1.
Targeting frequency for deletion vectors in embryonic stem cells.
Authors: Zhang H , Hasty P , Bradley A
Published: January 2012

We analyzed the gene targeting frequencies and recombination products generated by a series of replacement deletion vectors which target the hprt (hypoxanthine phosphoribosyltransferase) locus in mouse embryonic stem cells. We found that the targeting frequency of a 19.2-kb deletion was comparable to that of a 3-kb deletion or a conventional replacement event in which a 1.7-kb fragment was inserted into the locus. We also observed different integration patterns for these deletion vectors. A result of this finding is that a wide range of genomic deletions in embryonic stem cells is feasible.
Expression of the plasmodial pfmdr1 gene in mammalian cells is associated with increased susceptibility to chloroquine.
Authors: van Es H H , Karcz S , Chu F , Cowman A F , Vidal S , Gros P , Schurr E
Published: January 2012

Chloroquine (CQ)-resistant (CQR) Plasmodium falciparum malaria parasites show a strong decrease in CQ accumulation in comparison with chloroquine-sensitive parasites. Controversy exists over the role of the plasmodial pfmdr1 gene in the CQR phenotype. pfmdr1 is a member of the superfamily of ATP-binding cassette transporters. Other members of this family are the mammalian multidrug resistance genes and the CFTR gene. We have expressed the pfmdr1-encoded protein, Pgh1, in CHO cells and Xenopus oocytes. CHO cells expressing the Pgh1 protein demonstrated an increased, verapamil-insensitive susceptibility to CQ. Conversely, no increase in drug susceptibility to primaquine, quinine, adriamycin, or colchicine was observed in Pgh1-expressing cells. CQ uptake experiments revealed an increased, ATP-dependent accumulation of CQ in Pgh1-expressing cells over the level in nonexpressing control cells. The increased CQ accumulation in Pgh1-expressing cells coincided with an enhanced in vivo inhibition of lysosomal alpha-galactosidase by CQ. CHO cells expressing Pgh1 carrying two of the CQR-associated Pgh1 amino acid changes (S1034C and N1042D) did not display an increased CQ sensitivity. Immunofluorescence experiments revealed an intracellular localization of both mutant and wild-type forms of Pgh1. We conclude from our results that wild-type Pgh1 protein can mediate an increased intracellular accumulation of CQ and that this function is impaired in CQR-associated mutant forms of the protein. We speculate that the Pgh1 protein plays an important role in CQ import in CQ-sensitive malaria parasites.
rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling in KB cells.
Authors: Nishiyama T , Sasaki T , Takaishi K , Kato M , Yaku H , Araki K , Matsuura Y , Takai Y
Published: January 2012

Insulin and hepatocyte growth factor (HGF) induced morphologically different membrane rufflings in KB cells. Insulin-induced membrane ruffling was inhibited by microinjection of rho GDI, an inhibitory GDP/GTP exchange regulator for both rho p21 and rac p21 small GTP-binding proteins, but not inhibited by microinjection of botulinum exoenzyme C3, known to selectively ADP-ribosylate rho p21 and to impair its function. This rho GDI action was prevented by comicroinjection with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound rac1 p21. In contrast, HGF-induced membrane ruffling was inhibited by microinjection of rho GDI or C3. This rho GDI action was prevented by comicroinjection with GTP gamma S-bound rhoA p21, and this C3 action was prevented by comicroinjection with GTP gamma S-bound rhoAIle-41 p21, which is resistant to C3. Microinjection of either GTP gamma S-bound rac1 p21 or rhoA p21 alone induced membrane ruffling in the absence of the growth factors. The rac1 p21-induced membrane ruffling was morphologically similar to the insulin-induced kind, whereas rhoA p21-induced ruffling was apparently different from both the insulin- and HGF-induced kinds. Membrane ruffling was also induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, but not by Ca2+ ionophore or microinjection of a dominant active Ki-ras p21 mutant (Ki-rasVal-12 p21). The phorbol ester-induced membrane ruffling was morphologically similar to the rhoA p21-induced kind and inhibited by microinjection of rho GDI or C3. These results indicate that rac p21 and rho GDI are involved in insulin-induced membrane ruffling and that rho p21 and rho GDI are involved in HGF- and phorbol ester-induced membrane rufflings.
Identification of I-plastin, a human fimbrin isoform expressed in intestine and kidney.
Authors: Lin C S , Shen W , Chen Z P , Tu Y H , Matsudaira P
Published: January 2012

The complete cDNA sequence of human intestine-specific plastin (I-plastin) was determined from a clone derived by PCR. It consists of a 97-bp 5' untranslated region, a 1,887-bp coding region, and a 1,655-bp 3' untranslated region. The coding region predicts a 629-residue polypeptide whose sequence displays 86, 75, and 73% identities with chicken intestine fimbrin, human T-plastin, and human L-plastin, respectively. Recombinant I-plastin cross-linked actin filaments into bundles in the absence but not in the presence of calcium. The I-plastin gene was mapped by PCR to human chromosome 3; the L- and T-plastin genes were previously mapped to chromosomes 13 and X, respectively. I-plastin mRNA was detected in the small intestine, colon, and kidneys; relatively lower levels of expression were detected in the lungs and stomach. In contrast, L-plastin expression was restricted to the spleen and other lymph node-containing organs, while T-plastin was expressed in a variety of organs, including muscle, brain, uterus, and esophagus. In contrast to the situation for the intestine, high levels of L- and T-plastin mRNAs were detected in Caco-2, a human colon-derived cell line. Immunofluorescence microscopy detected I-plastin in the brush border of the small intestine and colon. These results identify I-plastin as the human homolog of chicken intestine fimbrin and as a third plastin isoform in humans.
Loss of p53 protein during radiation transformation of primary human mammary epithelial cells.
Authors: Wazer D E , Chu Q , Liu X L , Gao Q , Safaii H , Band V
Published: January 2012

The causative factors leading to breast cancer are largely unknown. Increased incidence of breast cancer following diagnostic or therapeutic radiation suggests that radiation may contribute to mammary oncogenesis. This report describes the in vitro neoplastic transformation of a normal human mammary epithelial cell strain, 76N, by fractionated gamma-irradiation at a clinically used dose (30 Gy). The transformed cells (76R-30) were immortal, had reduced growth factor requirements, and produced tumors in nude mice. Remarkably, the 76R-30 cells completely lacked the p53 tumor suppressor protein. Loss of p53 was due to deletion of the gene on one allele and a 26-bp deletion within the third intron on the second allele which resulted in abnormal splicing out of either the third or fourth exon from the mRNA. PCR with a mutation-specific primer showed that intron 3 mutation was present in irradiated cells before selection for immortal phenotype. 76R-30 cells did not exhibit G1 arrest in response to radiation, indicating a loss of p53-mediated function. Expression of the wild-type p53 gene in 76R-30 cells led to their growth inhibition. Thus, loss of p53 protein appears to have contributed to neoplastic transformation of these cells. This unique model should facilitate analyses of molecular mechanisms of radiation-induced breast cancer and allow identification of p53-regulated cellular genes in breast cells.
MCM1 point mutants deficient in expression of alpha-specific genes: residues important for interaction with alpha 1.
Authors: Bruhn L , Sprague G F
Published: January 2012

Complexes formed between MCM1 and several coregulatory proteins--alpha 1, alpha 2, and STE12--serve to govern transcription of the a- and alpha-specific gene sets in the yeast Saccharomyces cerevisiae. The N-terminal third of MCM1, MCM1(1-98), which includes a segment homologous to mammalian serum response factor, is capable of performing all of the functions necessary for cell-type-specific gene regulation, including DNA binding and interaction with coregulatory proteins. To explore the mechanisms by which MCM1(1-98) functions, we isolated point mutants that are specifically deficient in alpha-specific gene expression in vivo, anticipating that many of the mutants would be impaired for interaction with alpha 1. Indeed, in vitro DNA binding assays revealed that a substantial number of the mutants were specifically defective in the ability to bind cooperatively with alpha 1. Two other mutant classes were also found. One class, exemplified most clearly by substitutions at residues 22 and 27, exhibited a general defect in DNA binding. The second class, exemplified by substitutions at residues 33 and 41, was proficient at DNA binding and interaction with alpha 1 in vitro, suggesting that these mutants may be defective in achieving an alpha 1-mediated conformational change required for transcription activation in vivo. Most of the mutants defective for interaction with alpha 1 had substitutions within residues 69 to 81, which correspond to a region of serum response factor important for interaction with its coregulatory proteins. A subset of the mutants with changes in this region were also defective in the ability to bind with STE12 to DNA from an a-specific gene, suggesting that a common region of MCM1(1-98) mediates interaction with both alpha 1 and STE12. This region of MCM1 does not seem to constitute an independent domain of the protein, however, because some substitutions within this region affected DNA binding. Only two of the MCM1(1-98) point mutants showed significant defects in the ability to form complexes with alpha 2, suggesting that the mechanism by which MCM1 interacts with alpha 2 is distinct from that by which it interacts with alpha 1 and STE12.