bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of Ca
V
2.1 channels by Ca
2+
/calmodulin-dependent protein kinase II bound to the C-terminal domain
Authors: Jiang Xin , Lautermilch Nathan J. , Watari Hirofumi , Westenbroek Ruth E. , Scheuer Todd , Catterall William A.
Published: December 2007

Ca
2+
/calmodulin-dependent protein kinase II (CaMKII) is a key regulator of synaptic responses in the postsynaptic density, but understanding of its mechanisms of action in the presynaptic neuron is incomplete. Here we show that CaMKII constitutively associates with and modulates voltage-gated calcium (Ca
V
)2.1 channels that conduct P/Q type Ca
2+
currents and initiate transmitter release. Both exogenous and brain-specific inhibitors of CaMKII accelerate voltage-dependent inactivation, cause a negative shift in the voltage dependence of inactivation, and reduce Ca
2+
-dependent facilitation of Ca
V
2.1 channels. The modulatory effects of CaMKII are reduced by a peptide that prevents binding to Ca
V
2.1 channels but not by a peptide that blocks catalytic activity, suggesting that binding rather than phosphorylation is responsible for modulation. Our results reveal a signaling complex formed by Ca
V
2.1 channels and CaMKII that regulates P/Q-type Ca
2+
current in neurons. We propose an “effector checkpoint” model for the control of Ca
2+
channel fitness for function that depends on association with CaMKII, SNARE proteins, and other effectors of Ca
2+
signals. This regulatory mechanism would be important in presynaptic nerve terminals, where Ca
V
2.1 channels initiate synaptic transmission and CaMKII has noncatalytic effects on presynaptic plasticity.
Binding of rapamycin analogs to calcium channels and FKBP52 contributes to their neuroprotective activities
Authors: Ruan Benfang , Pong Kevin , Jow Flora , Bowlby Mark , Crozier Robert A. , Liu Danni , Liang Shi , Chen Yi , Mercado Mary Lynn , Feng Xidong , Bennett Frann , von Schack David , McDonald Leonard , Zaleska Margaret M. , Wood Andrew , Reinhart Peter H. , Magolda Ronald L. , Skotnicki Jerauld , Pangalos Menelas N. , Koehn Frank E. , Carter Guy T. , Abou-Gharbia Magid , Graziani Edmund I.
Published: December 2007

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the β1-subunit of L-type voltage-dependent Ca2+channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca2+channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca2+-induced cell death by modulating Ca2+channels and promote neurite outgrowth via FKBP52 binding.
Extracellular matrix production and calcium carbonate precipitation by coral cells
in vitro
Authors: Helman Yael , Natale Frank , Sherrell Robert M. , LaVigne Michèle , Starovoytov Valentin , Gorbunov Maxim Y. , Falkowski Paul G.
Published: December 2007

The evolution of multicellularity in animals required the production of extracellular matrices that serve to spatially organize cells according to function. In corals, three matrices are involved in spatial organization: (
i
) an organic ECM, which facilitates cell–cell and cell–substrate adhesion; (
ii
) a skeletal organic matrix (SOM), which facilitates controlled deposition of a calcium carbonate skeleton; and (
iii
) the calcium carbonate skeleton itself, which provides the structural support for the 3D organization of coral colonies. In this report, we examine the production of these three matrices by using an
in vitro
culturing system for coral cells. In this system, which significantly facilitates studies of coral cell physiology, we demonstrate
in vitro
excretion of ECM by primary (nondividing) tissue cultures of both soft (
Xenia elongata
) and hard (
Montipora digitata
) corals. There are structural differences between the ECM produced by
X. elongata
cell cultures and that of
M. digitata
, and ascorbic acid, a critical cofactor for proline hydroxylation, significantly increased the production of collagen in the ECM of the latter species. We further demonstrate
in vitro
production of SOM and extracellular mineralized particles in cell cultures of
M. digitata
. Inductively coupled plasma mass spectrometry analysis of Sr/Ca ratios revealed the particles to be aragonite.
De novo
calcification was confirmed by following the incorporation of
45
Ca into acid labile macromolecules. Our results demonstrate the ability of isolated, differentiated coral cells to undergo fundamental processes required for multicellular organization.
The Parkinson's disease protein α-synuclein disrupts cellular Rab homeostasis
Authors: Gitler Aaron D. , Bevis Brooke J. , Shorter James , Strathearn Katherine E. , Hamamichi Shusei , Su Linhui Julie , Caldwell Kim A. , Caldwell Guy A. , Rochet Jean-Christophe , McCaffery J. Michael , Barlowe Charles , Lindquist Susan
Published: December 2007

α-Synuclein (α-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinson's disease (PD). In yeast α-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER→Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER→Golgi trafficking. The homologous Rab1 rescues α-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of α-syn pathobiology. In a cell-free system with purified transport factors α-syn inhibited ER→Golgi trafficking in an α-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the
in vivo
trafficking problem is due to a direct effect of α-syn on the transport machinery. By ultrastructural analysis the earliest
in vivo
defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with α-syn and with diverse vesicle markers, suggesting that α-syn can impair multiple trafficking steps. Other Rabs did not ameliorate α-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, α-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive.
High-resolution mapping of DNA hypermethylation and hypomethylation in lung cancer
Authors: Rauch Tibor A. , Zhong Xueyan , Wu Xiwei , Wang Melody , Kernstine Kemp H. , Wang Zunde , Riggs Arthur D. , Pfeifer Gerd P.
Published: December 2007

Changes in DNA methylation patterns are an important characteristic of human cancer. Tumors have reduced levels of genomic DNA methylation and contain hypermethylated CpG islands, but the full extent and sequence context of DNA hypomethylation and hypermethylation is unknown. Here, we used methylated CpG island recovery assay-assisted high-resolution genomic tiling and CpG island arrays to analyze methylation patterns in lung squamous cell carcinomas and matched normal lung tissue. Normal tissues from different individuals showed overall very similar DNA methylation patterns. Each tumor contained several hundred hypermethylated CpG islands. We identified and confirmed 11 CpG islands that were methylated in 80–100% of the SCC tumors, and many hold promise as effective biomarkers for early detection of lung cancer. In addition, we find that extensive DNA hypomethylation in tumors occurs specifically at repetitive sequences, including short and long interspersed nuclear elements and LTR elements, segmental duplications, and subtelomeric regions, but single-copy sequences rarely become demethylated. The results are consistent with a specific defect in methylation of repetitive DNA sequences in human cancer.
High numerical aperture tabletop soft x-ray diffraction microscopy with 70-nm resolution
Authors: Sandberg Richard L. , Song Changyong , Wachulak Przemyslaw W. , Raymondson Daisy A. , Paul Ariel , Amirbekian Bagrat , Lee Edwin , Sakdinawat Anne E. , La-O-Vorakiat Chan , Marconi Mario C. , Menoni Carmen S. , Murnane Margaret M. , Rocca Jorge J. , Kapteyn Henry C. , Miao Jianwei
Published: December 2007

Light microscopy has greatly advanced our understanding of nature. The achievable resolution, however, is limited by optical wavelengths to ≈200 nm. By using imaging and labeling technologies, resolutions beyond the diffraction limit can be achieved for specialized specimens with techniques such as near-field scanning optical microscopy, stimulated emission depletion microscopy, and photoactivated localization microscopy. Here, we report a versatile soft x-ray diffraction microscope with 70- to 90-nm resolution by using two different tabletop coherent soft x-ray sources—a soft x-ray laser and a high-harmonic source. We also use field curvature correction that allows high numerical aperture imaging and near-diffraction-limited resolution of 1.5λ. A tabletop soft x-ray diffraction microscope should find broad applications in biology, nanoscience, and materials science because of its simple optical design, high resolution, large depth of field, 3D imaging capability, scalability to shorter wavelengths, and ultrafast temporal resolution.
Risk of Hand or Glove Contamination After Contact With Patients Colonized With Vancomycin-ResistantEnterococcusor the Colonized Patients' Environment
Authors: Hayden Mary K. , Blom Donald W. , Lyle Elizabeth A. , Moore Charity G. , Weinstein Robert A.
Published: December 2007

Objective.

To estimate the level of hand or glove contamination with vancomycin-resistant enterococci (VRE) among healthcare workers (HCWs) who touch a patient colonized with VRE and/or the colonized patient's environment during routine care.

Design.

Structured observational study.

Setting.

Medical intensive care unit of a 700-bed, tertiary-care teaching hospital.

Participants.

VRE-colonized patients and their caregivers.

Methods.

We obtained samples from sites on the intact skin of 22 patients colonized with VRE and samples from sites in the patients' rooms, before and after routine care, during 27 monitoring episodes. A total of 98 unique HCWs were observed during 131 HCW observations. Observers recorded the sites touched by HCWs. Culture samples were obtained from HCWs' hands and gloves before and after care.

Results.

VRE were isolated from a mean (±SD) of 55% ± 24% of patient sites (n= 256) and 17% ± 12% of environmental sites (n= 1,572). Most HCWs (131 [56%]) touched both the patient and the patient's environment; no HCW touched only the patient. Of 103 HCWs whose hand samples were negative for VRE when they entered the room, 52% contaminated their hands or gloves after touching the environment, and 70% contaminated their hands or gloves after touching the patient and the environment (P= .101). In a univariate logistic regression model, the risk of hand or glove contamination was associated with the number of contacts made (odds ratio, 1.1 [95% confidence interval, 1.01-1.19). In a multivariate model, the effect of the number of contacts could not be distinguished from the effect of type of contact (ie, touching the environment alone or touching both the patient and the environment). Overall, 37% of HCWs who did not wear gloves contaminated their hands, and 5% of HCWs who wore gloves did so (an 86% difference).

Conclusion.

HCWs were nearly as likely to have contaminated their hands or gloves after touching the environment in a room occupied by a patient colonized by VRE as after touching the colonized patient and the patient's environment. Gloves were highly protective with respect to hand contamination.
Influenza Vaccination Among Registered Nurses: Information Receipt, Knowledge, and Decision-Making at an Institution With a Multifaceted Educational Program
Authors: Ofstead Cori L. , Tucker Sharon J. , Beebe Timothy J. , Poland Gregory A.
Published: December 2007

Objective.

To evaluate the receipt of information and knowledge about influenza and vaccination, as well as influenza vaccination status and reasons for declining vaccination, among registered nurses.

Design.

Cross-sectional survey of registered nurses (RNs).

Setting.

A large tertiary medical center with a long-standing, multifaceted influenza vaccination program and relatively high vaccination rates among employees overall (76.5%).

Participants.

Randomly selected group of 990 RNs employed as inpatient staff nurses at the institution.

Results.

The survey was completed by 513 (51.8%) of 990 RNs. Most RNs (86.7%) had received an influenza vaccination in the past, and 331 (64.5%) intended to receive vaccination during the 2005-2006 influenza season. More than 90% of RNs acknowledged exposure to educational bulletins, and most had received information about influenza severity (383 [74.7%]), transmission (398 [77.6%]), vaccine safety (416 [81.1%]), and the time and location of free vaccination (460 [89.7%]). A majority (436 [85.0%]) felt they had received all the information they needed to make good decisions about vaccination. However, only 49 RNs (9.6%) gave correct answers to more than 85% of the knowledge questions on the survey. The reasons most frequently reported for declining vaccination were doubts about the risk of influenza and the need for vaccination, concerns about vaccine effectiveness and side effects, and dislike of injections.

Conclusions.

RNs exposed to a longstanding, multifaceted educational program had received information about influenza vaccination, but misconceptions were common and only 331 (64.5%) intended to receive vaccination. Strategies other than educational interventions are needed to increase influenza vaccination rates and thereby to ensure healthcare worker and patient safety.
Receptivity to Mandatory Influenza Vaccination Policies for Healthcare Workers Among Registered Nurses Working on Inpatient Units
Authors: Poland Gregory A. , Ofstead Cori L. , Tucker Sharon J. , Beebe Timothy J.
Published: December 2007

A survey that included questions about preferred methods of influenza prevention was completed by 513 registered nurses working on inpatient units. Vaccination was the preferred influenza prevention method among 83.0% of respondents. Of 506 respondents, 283 (56.0%) stated that mandatory influenza vaccination was appropriate for healthcare workers, and 394 (59.4%) of 512 RNs reported that they would support a policy requiring annual influenza vaccination for healthcare workers that allowed for informed declination.
Use of a Pandemic Preparedness Drill to Increase Rates of Influenza Vaccination Among Healthcare Workers
Authors: Kuntz Jennifer L. , Holley Stephanie , Helms Charles M. , Cavanaugh Joseph E. , Berg Jeff Vande , Herwaldt Loreen A. , Polgreen Philip M.
Published: December 2007

Objective.

To determine the effect of a pandemic influenza preparedness drill on the rate of influenza vaccination among healthcare workers (HCWs).

Design.

Before-after intervention trial.

Setting.

The University of Iowa Hospitals and Clinics (UIHC), a large, academic medical center, during 2005.

Subjects.

Staff members at UIHC.

Methods.

UIHC conducted a pandemic influenza preparedness drill that included a goal of vaccinating a large number of HCWs in 6 days without disrupting patient care. Peer vaccination and mobile vaccination teams were used to vaccinate HCWs, educational tools were distributed to encourage HCWs to be vaccinated, and resources were allocated on the basis of daily vaccination reports. Logit models were used to compare vaccination rates achieved during the 2005 vaccination drill with the vaccination rates achieved during the 2003 vaccination campaign.

Results.

UIHC vaccinated 54% of HCWs (2,934 of 5,467) who provided direct patient care in 6 days. In 2 additional weeks, this rate increased to 66% (3,625 of 5,467). Overall, 66% of resident physicians (311 of 470) and 63% of nursing staff (1,429 of 2,255) were vaccinated. Vaccination rates in 2005 were significantly higher than the hospitalwide rate of 41% (5,741 of 14, 086) in 2003.

Conclusions.

UIHC dramatically increased the influenza vaccination rate among HCWs by conducting a pandemic influenza preparedness drill. Additionally, the drill allowed us to conduct a bioemergency drill in a realistic scenario, use innovative methods for vaccine delivery, and secure administrative support for future influenza vaccination campaigns. Our study demonstrates how a drill can be used to improve vaccination rates significantly.
Importance of Bacterial Burden Among Methicillin-Resistant Staphylococcus aureus Carriers in a Long-Term Care Facility
Authors: Stone Nimalie D. , Lewis Donna R. , Lowery H. K. , Darrow Lyndsey A. , Kroll Catherine M. , Gaynes Robert P. , Jernigan John A. , McGowan John E. , Tenover Fred C. , Richards Chesley L.
Published: December 2007

Objective.

To evaluate the prevalence and transmission of methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization, as well as risk factors associated with MRSA carriage, among residents of a long-term care facility (LTCF).

Design.

Prospective, longitudinal cohort study.

Setting.

A 100-bed Veterans Administration LTCF

Participants.

All current and newly admitted residents of the LTCF during an 8-week study period.

Methods.

Nasal swab samples were obtained weekly and cultured on MRSA-selective media, and the cultures were graded for growth on a semiquantitative scale from 0 (no growth) to 6 (heavy growth). Epidemiologic data for the periods before and during the study were collected to assess risk factors for MRSA carriage.

Results.

Of 83 LTCF residents, 49 (59%) had 1 or more nasal swab cultures that were positive for MRSA; 34 (41%) were consistently culture-negative (designated “noncarriers”). Of the 49 culture-positive residents, 30 (36% of the total of 83 residents) had all cultures positive for MRSA (designated “persistent carriers”), and 19 (23% of the 83 residents) had at least 1 culture, but not all cultures, positive for MRSA (designated “intermittent carriers”). Multivariate analysis showed that participants with at least 1 nasal swab culture positive for MRSA were likely to have had previous hospitalization (odds ratio, 3.9) or wounds (odds ratio, 8.2). Persistent carriers and intermittent carriers did not differ in epidemiologic characteristics but did differ in mean MRSA growth score (3.7 vs 0.7; P < .001).

Conclusions.

Epidemiologic characteristics differed between noncarriers and subjects with at least 1 nasal swab culture positive for MRSA. However, in this LTCF population, only the degree of bacterial colonization (as reflected by mean MRSA growth score) distinguished persistent carriers from intermittent carriers. Understanding the burden of colonization may be important when determining future surveillance and control strategies.
Clinical and Economic Consequences of Failure of Initial Antibiotic Therapy for Hospitalized Patients With Complicated Skin and Skin-Structure Infections
Authors: Edelsberg John , Berger Ariel , Weber David J. , Mallick Rajiv , Kuznik Andreas , Oster Gerry
Published: December 2007

Objective.

To estimate the consequences of failure of initial antibiotic therapy for patients with complicated skin and skin-structure infections.

Design.

Retrospective cohort study.

Setting.

Large US multihospital database.

Patients.

We identified a total of 47,219 patients (age 18 years or older) who were admitted to the hospital for complicated skin and skin-structure infections from April 1, 2003, through March 31, 2004, and who received intravenous antibiotics during the first 2 hospital-days (ie, initial antibiotic therapy). Failure of therapy was defined as drainage, debridement, or receipt of other intravenous antibiotics at any subsequent time (except for changes to narrower-spectrum agents or any therapy change immediately before discharge). Predictors of failure of antibiotic therapy and mortality were examined using multivariate logistic regression. Analysis of covariance was used to estimate the impact of treatment failure on duration of intravenous antibiotic therapy, length of stay, and total inpatient charges.

Results.

For 10,782 admitted patients (22.8%), there was evidence of failure of initial antibiotic therapy. In multivariate analyses, treatment failure was associated with receipt of vasoactive medications during the first 2 hospital-days (odds ratio [OR], 1.66 [95% confidence interval {CI}, 1.19-2.31]), initiation of antibiotic therapy in the intensive care unit (OR, 1.53 [95% CI, 1.28-1.84]), and the patient's Charlson comorbidity index (OR per 1-point increase, 1.06 [95% CI, 1.04-1.08]); treatment failure was also was associated with a 3-fold increase in mortality (OR, 2.91 [95% CI, 2.34-3.62]). Compared with patients for whom initial treatment was successful, patients who experienced treatment failure received intravenous antibiotic therapy for a mean of 5.7 additional days, were hospitalized for a mean of 5.4 additional days, and incurred a mean of $5,285 (in 2003 dollars) in additional inpatient charges (all P <.01).

Conclusion.

Failure of initial antibiotic therapy in the treatment of complicated skin and skin-structure infections is associated with significantly worse clinical and economic outcomes.
House Staff Attitudes About Influenza Vaccination After Participation in a Clinical Trial to Improve Vaccination of Hospital Patients
Authors: Cabello Javier , Das Krishna , Trick William E. , Gerard Mary N. , Charles-Damte Marjorie
Published: December 2007

We surveyed house staff who had participated in a trial that compared influenza vaccination strategies for inpatients. House staff who were exposed to computer-generated vaccination orders were more likely to report that they recommended vaccination to their inpatients and outpatients, compared with house staff who were not exposed to a vaccination intervention. Also, house staff did not recognize regnant women as a high-priority population for influenza vaccination.
Patient Attitudes Regarding Participation in Studies of Antimicrobial Resistance
Authors: Tolomeo Pam , Wheeler Mary , Metlay Joshua P. , Armstrong Katrina , Fishman Neil O. , Bilker Warren B. , Lautenbach Ebbing
Published: December 2007

Background.

A number of recent studies of antimicrobial resistance have focused on the role of antimicrobial-resistant pathogens that colonize the gastrointestinal tract. However, participation rates have been low in studies that involve fecal sampling. Attitudes toward such studies among potential study participants have not been assessed.

Methods.

We conducted a cross-sectional survey, enrolling 3 groups of inpatients from a large academic center. Group 1 consisted of patients who had previously participated in a cohort study of fluoroquinolone-resistant Escherichia coli, which involved the collection of perirectal swab samples. Group 2 consisted of patients who had previously refused to participate in the study of fluoroquinolone-resistant E. coli. Group 3 consisted of patients who had never been asked to participate in the study of the fluoroquinolone-resistant E. coli. The survey assessed patients' attitudes and beliefs regarding medical research and their willingness to consent to collection of a perirectal swab sample. Response options were recorded on a 5-point Likert scale. The Fisher exact test was used to compare dichotomized responses across study groups.

Results.

A total of 90 patients were surveyed: there were 29 in group 1 and in group 2 and 32 in group 3. Of 90 patients, 31 (35%) believed researchers might run additional tests on collected samples without informing the patient, whereas 25 (27%) believed persons other than the research team might gain access to study results. The belief that a person could get sicker as a result of a having a perirectal swab sample collected was significantly more common among patients who had previously refused to participate in the fluoroquinolone-resistant E. coli study.

Conclusion.

This study highlights important beliefs and attitudes that are associated with the likelihood of participating in studies of antimicrobial resistance. Explicitly addressing these concerns with eligible patients is critical to optimize participation in future studies.
Macrolide and Clindamycin Resistance in Staphylococcus aureus Isolates and Antibiotic Use in a Veterans Affairs Medical Center
Authors: Modak Rohit , Ross David , Kan Virginia L.
Published: December 2007

A retrospective analysis of 13,946 Staphylococcus aureus isolates revealed a stable incidence of isolates that were resistant to both clindamycin and erythromycin, but a significantly increasing incidence of isolates that were susceptible to clindamycin and resistant to erythromycin during 1991-1995, 1996-2000, and 2001-2005. The use of macrolides and clindamycin also increased during 1996-2005. The incidence of S. aureus isolates with inducible clindamycin resistance increased steadily and significantly during the period from August 2004 through December 2005.
StARD13(Dlc-2) RhoGap Mediates Ceramide Activation of Phosphatidylglycerolphosphate Synthase and Drug Response in Chinese Hamster Ovary Cells
Authors: Hatch Grant M. , Gu Yuan , Xu Fred Y. , Cizeau Jeannick , Neumann Shannon , Park Ji-Seon , Loewen Shauna , Mowat Michael R.A.
Published: December 2007

To identify genes involved in etoposide drug response, we used promoter trap mutagenesis to isolate an etoposide-resistant Chinese hamster ovary (CHO) cell line. This resistant CHO-K1 line, named E91, showed cross-resistance to C2-ceramide (N-acetylsphingosine). The promoter trap retrovirus was found integrated into intron 1–2 of the Dlc-2 (Stard13) RhoGap gene. The E91 cells showed elevated guanosine triphosphate (GTP)-bound RhoA levels compared with the parental line, suggesting that retrovirus integration had inactivated one of the Dlc-2 RhoGap alleles. To test whether E91 cells were impaired in an intracellular ceramide-regulated process not directly related to cell killing, we measured mitochondrial phosphatidylglycerolphosphate (PGP) synthase and phospholipase A2 enzyme activities in cells after C2-ceramide addition. Parental cells showed elevated enzyme activities after treatment with C2-ceramide or tumor necrosis factor α, but not the E91 cells. These results suggested that intracellular ceramide signaling was defective in E91 cells due to increased levels of active GTP-bound RhoA. RNA knockdown experiments of the Dlc2 RhoGap resulted in increased GTP-bound RhoA and reduced induction of PGP synthase after C2-ceramide addition compared with controls. Expression of a dominant-negative RhoA in the E91 cell line allowed induction of PGP synthase by ceramide. The RNA interference knockdown cell line also showed increased etoposide resistance. This study is the first report for the regulation of a phospholipid biosynthetic enzyme through RhoGap expression.
The Same Receptor, G Protein, and Mitogen-activated Protein Kinase Pathway Activate Different Downstream Regulators in the Alternative White and Opaque Pheromone Responses ofCandida albicans
Authors: Yi Song , Sahni Nidhi , Daniels Karla J. , Pujol Claude , Srikantha Thyagarajan , Soll David R.
Published: December 2007

Candida albicans must undergo a switch from white to opaque to mate. Opaque cells then release mating type-specific pheromones that induce mating responses in opaque cells. Uniquely in C. albicans, the same pheromones induce mating-incompetent white cells to become cohesive, form an adhesive basal layer of cells on a surface, and then generate a thicker biofilm that, in vitro, facilitates mating between minority opaque cells. Through mutant analysis, it is demonstrated that the pathways regulating the white and opaque cell responses to the same pheromone share the same upstream components, including receptors, heterotrimeric G protein, and mitogen-activated protein kinase cascade, but they use different downstream transcription factors that regulate the expression of genes specific to the alternative responses. This configuration, although common in higher, multicellular systems, is not common in fungi, and it has not been reported in Saccharomyces cerevisiae. The implications in the evolution of multicellularity in higher eukaryotes are discussed.
The DEAD-Box RNA Helicase Ded1p Affects and Accumulates inSaccharomyces cerevisiaeP-Bodies
Authors: Beckham Carla , Hilliker Angela , Cziko Anne-Marie , Noueiry Amine , Ramaswami Mani , Parker Roy
Published: December 2007

Recent results suggest that cytoplasmic mRNAs can form translationally repressed messenger ribonucleoprotein particles (mRNPs) capable of decapping and degradation, or accumulation into cytoplasmic processing bodies (P-bodies), which can function as sites of mRNA storage. The proteins that function in transitions between the translationally repressed mRNPs that accumulate in P-bodies and mRNPs engaged in translation are largely unknown. Herein, we demonstrate that the yeast translation initiation factor Ded1p can localize to P-bodies. Moreover, depletion of Ded1p leads to defects in P-body formation. Overexpression of Ded1p results in increased size and number of P-bodies and inhibition of growth in a manner partially suppressed by loss of Pat1p, Dhh1p, or Lsm1p. Mutations that inactivate the ATPase activity of Ded1p increase the overexpression growth inhibition of Ded1p and prevent Ded1p from localizing in P-bodies. Combined with earlier work showing Ded1p can have a positive effect on translation, these results suggest that Ded1p is a bifunctional protein that can affect both translation initiation and P-body formation.
Relative Structural and Functional Roles of Multiple Deubiquitylating Proteins Associated with Mammalian 26S Proteasome
Authors: Koulich Elena , Li Xiaohua , DeMartino George N.
Published: December 2007

We determined composition and relative roles of deubiquitylating proteins associated with the 26S proteasome in mammalian cells. Three deubiquitylating activities were associated with the 26S proteasome: two from constituent subunits, Rpn11/S13 and Uch37, and one from a reversibly associated protein, Usp14. RNA interference (RNAi) of Rpn11/S13 inhibited cell growth, decreased cellular proteasome activity via disrupted 26S proteasome assembly, and inhibited cellular protein degradation. In contrast, RNAi of Uch37 or Usp14 had no detectable effect on cell growth, proteasome structure or proteolytic capacity, but accelerated cellular protein degradation. RNAi of both Uch37 and Usp14 also had no effect on proteasome structure or proteolytic capacity, but inhibited cellular protein degradation. Thus, proper proteasomal processing of ubiquitylated substrates requires Rpn11 plus either Uch37 or Usp14. Although the latter proteins feature redundant deubiquitylation functions, they also appear to exert noncatalyic effects on proteasome activity that are similar to but independent of one another. These results reveal unexpected functional relationships among multiple deubiquitylating proteins and suggest a model for mammalian 26S proteasome function whereby their concerted action governs proteasome function by linking deubiquitylation to substrate hydrolysis.
Catalytic Therapy of Cancer with Ascorbate and Extracts of Medicinal Herbs
Authors: Rozanova (Torshina) Nadejda , Zhang Jin Z. , Heck Diane E.
Published: December 2007

Catalytic therapy (CT) is a cancer treatment modality based on the generation of reactive oxygen species (ROS) using a combination of substrate molecules and a catalyst. The most frequently used substrate/catalyst pair is ascorbate/Co phthalocyanine (PcCo). In the present work, herb extracts containing pigments have been studied as a catalyst in place of PcCo. Extracts from herbs are expected to have efficiency comparable with that of phthalocyanines but as natural products, to exhibit fewer side effects. The present studies demonstrate that a combined use of ascorbate and herbal extracts results in ROS production and a significant decrease in the number of cancer cells after a single in vitro treatment. Treatment with ascorbate in conjunction with extracts prepared from several medicinal herbs stimulated apoptosis and disrupted the cell cycle. The number of cells accumulating in the sub‐G0/G1 stage of the cell cycle was increased 2‐ to 7‐fold, and cells in G2/M increased 1.5‐ to 20‐fold, indicating that the treatment protocol was highly effective in suppressing DNA synthesis and potentially reflecting DNA damage in the tumor cells. In addition, 20–40% of the cells underwent apoptosis within 24 h of completing treatment. Our results suggest that herbal extracts can function as CT catalysts in the treatment of cancer.
Comfrey (Symphytum officinale. L.) and Experimental Hepatic Carcinogenesis: A Short‐Term Carcinogenesis Model Study
Authors: Gomes Maria Fernanda Pereira Lavieri , de Oliveira Massoco Cristina , Xavier José Guilherme , Bonamin Leoni Villano
Published: December 2007

Comfrey or Symphytum officinale (L.) (Boraginaceae) is a very popular plant used for therapeutic purposes. Since the 1980s, its effects have been studied in long‐term carcinogenesis studies, in which Comfrey extract is administered at high doses during several months and the neoplastic hepatic lesions are evaluated. However, the literature on this topic is very poor considering the studies performed under short‐term carcinogenesis protocols, such as the ‘resistant hepatocyte model’ (RHM). In these studies, it is possible to observe easily the phenomena related to the early phases of tumor development, since pre‐neoplastic lesions (PNLs) rise in about 1–2 months of chemical induction. Herein, the effects of chronic oral treatment of rats with 10% Comfrey ethanolic extract were evaluated in a RHM. Wistar rats were sequentially treated with N‐nitrosodiethylamine (ip) and 2‐acetilaminofluorene (po), and submitted to hepatectomy to induce carcinogenesis promotion. Macroscopic/microscopic quantitative analysis of PNL was performed. Non‐parametric statistical tests (Mann–Whitney and χ2) were used, and the level of significance was set at P ≤ 0.05. Comfrey treatment reduced the number of pre‐neoplastic macroscopic lesions up to 1 mm (P ≤ 0.05), the percentage of oval cells (P = 0.0001) and mitotic figures (P = 0.007), as well as the number of Proliferating Cell Nuclear Antigen (PCNA) positive cells (P = 0.0001) and acidophilic pre‐neoplastic nodules (P = 0.05). On the other hand, the percentage of cells presenting megalocytosis (P = 0.0001) and vacuolar degeneration (P = 0.0001) was increased. Scores of fibrosis, glycogen stores and the number of nucleolus organizing regions were not altered. The study indicated that oral treatment of rats with 10% Comfrey alcoholic extract reduced cell proliferation in this model.
GeBP and GeBP-Like Proteins Are Noncanonical Leucine-Zipper Transcription Factors That Regulate Cytokinin Response in Arabidopsis
Authors: Chevalier Florian , Perazza Daniel , Laporte Frédéric , Le Hénanff Gaëlle , Hornitschek Patricia , Bonneville Jean-Marc , Herzog Michel , Vachon Gilles
Published: December 2007

Understanding the role of transcription factors (TFs) is essential in reconstructing developmental regulatory networks. The plant-specific GeBP TF family of Arabidopsis thaliana (Arabidopsis) comprises 21 members, all of unknown function. A subset of four members, the founding member GeBP and GeBP-like proteins (GPL) 1, 2, and 3, shares a conserved C-terminal domain. Here we report that GeBP/GPL genes represent a newly defined class of leucine-zipper (Leu-zipper) TFs and that they play a redundant role in cytokinin hormone pathway regulation. Specifically, we demonstrate using yeast, in vitro, and split-yellow fluorescent protein in planta assays that GeBP/GPL proteins form homo- and heterodimers through a noncanonical Leu-zipper motif located in the C-terminal domain. A triple loss-of-function mutant of the three most closely related genes gebp gpl1 gpl2 shows a reduced sensitivity to exogenous cytokinins in a subset of cytokinin responses such as senescence and growth, whereas root inhibition is not affected. We find that transcript levels of type-A cytokinin response genes, which are involved in the negative feedback regulation of cytokinin signaling, are higher in the triple mutant. Using a GPL version that acts as a constitutive transcriptional activator, we show that the regulation of Arabidopsis response regulators (ARRs) is mediated by at least one additional, as yet unknown, repressor acting genetically downstream in the GeBP/GPL pathway. Our results indicate that GeBP/GPL genes encode a new class of unconventional Leu-zipper TF proteins and suggest that their role in the cytokinin pathway is to antagonize the negative feedback regulation on ARR genes to trigger the cytokinin response.
PSY3, a New Member of the Phytoene Synthase Gene Family Conserved in the Poaceae and Regulator of Abiotic Stress-Induced Root Carotenogenesis
Authors: Li Faqiang , Vallabhaneni Ratnakar , Wurtzel Eleanore T.
Published: December 2007

Abscisic acid (ABA) plays a vital role in mediating abiotic stress responses in plants. De novo ABA biosynthesis involves cleavage of carotenoid precursors by 9-cis-epoxycarotenoid dioxygenase (NCED), which is rate controlling in leaves and roots; however, additional bottlenecks in roots must be overcome, such as biosynthesis of upstream carotenoid precursors. Phytoene synthase (PSY) mediates the first committed step in carotenoid biosynthesis; with PSY3 described here, maize (Zea mays) and other members of the Poaceae have three paralogous genes, in contrast to only one in Arabidopsis thaliana. PSY gene duplication has led to subfunctionalization, with each paralog exhibiting differential gene expression. We showed that PSY3 encodes a functional enzyme for which maize transcript levels are regulated in response to abiotic stresses, drought, salt, and ABA. Drought-stressed roots showed elevated PSY3 transcripts and ABA, responses reversed by rehydration. By blocking root carotenoid biosynthesis with the maize y9 mutation, we demonstrated that PSY3 mRNA elevation correlates with carotenoid accumulation and that blocking carotenoid biosynthesis interferes with stress-induced ABA accumulation. In parallel, we observed elevated NCED transcripts and showed that, in contrast to dicots, root zeaxanthin epoxidase transcripts were unchanged. PSY3 was the only paralog for which transcripts were induced in roots and abiotic stress also affected leaf PSY2 transcript levels; PSY1 mRNA was not elevated in any tissues tested. Our results suggest that PSY3 expression influences root carotenogenesis and defines a potential bottleneck upstream of NCED; further examination of PSY3 in the grasses is of value for better understanding root-specific stress responses that impact plant yield.
Quantitative Conversion of Phytate to Inorganic Phosphorus in Soybean Seeds Expressing a Bacterial Phytase
Authors: Bilyeu Kristin D. , Zeng Peiyu , Coello Patricia , Zhang Zhanyuan J. , Krishnan Hari B. , Bailey April , Beuselinck Paul R. , Polacco Joe C.
Published: December 2007

Phytic acid (PA) contains the major portion of the phosphorus in the soybean (Glycine max) seed and chelates divalent cations. During germination, both minerals and phosphate are released upon phytase-catalyzed degradation of PA. We generated a soybean line (CAPPA) in which an Escherichia coli periplasmic phytase, the product of the appA gene, was expressed in the cytoplasm of developing cotyledons. CAPPA exhibited high levels of phytase expression, ≥90% reduction in seed PA, and concomitant increases in total free phosphate. These traits were stable, and, although resulted in a trend for reduced emergence and a statistically significant reduction in germination rates, had no effect on the number of seeds per plant or seed weight. Because phytate is not digested by monogastric animals, untreated soymeal does not provide monogastrics with sufficient phosphorus and minerals, and PA in the waste stream leads to phosphorus runoff. The expression of a cytoplasmic phytase in the CAPPA line therefore improves phosphorus availability and surpasses gains achieved by other reported transgenic and mutational strategies by combining in seeds both high phytase expression and significant increases in available phosphorus. Thus, in addition to its value as a high-phosphate meal source, soymeal from CAPPA could be used to convert PA of admixed meals, such as cornmeal, directly to utilizable inorganic phosphorus.
Functional Coexpression of the Mitochondrial Alternative Oxidase and Uncoupling Protein Underlies Thermoregulation in the Thermogenic Florets of Skunk Cabbage
Authors: Onda Yoshihiko , Kato Yoshiaki , Abe Yukie , Ito Takanori , Morohashi Miyuki , Ito Yuka , Ichikawa Megumi , Matsukawa Kazushige , Kakizaki Yusuke , Koiwa Hiroyuki , Ito Kikukatsu
Published: December 2007

Two distinct mitochondrial energy dissipating systems, alternative oxidase (AOX) and uncoupling protein (UCP), have been implicated as crucial components of thermogenesis in plants and animals, respectively. To further clarify the physiological roles of AOX and UCP during homeothermic heat production in the thermogenic skunk cabbage (Symplocarpus renifolius), we identified the thermogenic cells and performed expression and functional analyses of these genes in this organism. Thermographic analysis combined with in situ hybridization revealed that the putative thermogenic cells surround the stamens in the florets of skunk cabbage and coexpress transcripts for SrAOX, encoding Symplocarpus AOX, and SrUCPb, encoding a novel UCP that lacks a fifth transmembrane segment. Mitochondria isolated from the thermogenic florets exhibited substantial linoleic acid (LA)-inducible uncoupling activities. Moreover, our results demonstrate that LA is capable of inhibiting the mitochondrial AOX pathway, whereas the proportion of pyruvate-stimulated AOX capacity was not significantly affected by LA. Intriguingly, the protein expression levels for SrAOX and SrUCPb were unaffected even when the ambient air temperatures increased from 10.3°C to 23.1°C or from 8.3°C to 24.9°C. Thus, our results suggest that functional coexpression of AOX and UCP underlies the molecular basis of heat production, and that posttranslational modifications of these proteins play a crucial role in regulating homeothermic heat production under conditions of natural ambient temperature fluctuations in skunk cabbage.
The NADPH-Dependent Thioredoxin Reductase/Thioredoxin System in Germinating Barley Seeds: Gene Expression, Protein Profiles, and Interactions between Isoforms of Thioredoxin h and Thioredoxin Reductase
Authors: Shahpiri Azar , Svensson Birte , Finnie Christine
Published: December 2007

The NADPH-dependent thioredoxin reductase (NTR)/thioredoxin (Trx) system catalyzes disulfide bond reduction in the cytoplasm and mitochondrion. Trx h is suggested to play an important role in seed development, germination, and seedling growth. Plants have multiple isoforms of Trx h and NTR; however, little is known about the roles of the individual isoforms. Trx h isoforms from barley (Hordeum vulgare) seeds (HvTrxh1 and HvTrxh2) were characterized previously. In this study, two NTR isoforms (HvNTR1 and HvNTR2) were identified, enabling comparison of gene expression, protein appearance, and interaction between individual NTR and Trx h isoforms in barley embryo and aleurone layers. Although mRNA encoding both Trx h isoforms is present in embryo and aleurone layers, the corresponding proteins differed in spatiotemporal appearance. HvNTR2, but not HvNTR1, gene expression seems to be regulated by gibberellic acid. Recombinant HvNTR1 and HvNTR2 exhibited virtually the same affinity toward HvTrxh1 and HvTrxh2, whereas HvNTR2 has slightly higher catalytic activity than HvNTR1 with both Trx h isoforms, and HvNTR1 has slightly higher catalytic activity toward HvTrxh1 than HvTrxh2. Notably, both NTRs reduced Trx h at the acidic conditions residing in the starchy endosperm during germination. Interspecies reactions between the barley proteins and Escherichia coli Trx or Arabidopsis thaliana NTR, respectively, occurred with 20- to 90-fold weaker affinity. This first investigation of regulation and interactions between members of the NTR/Trx system in barley seed tissues suggests that different isoforms are differentially regulated but may have overlapping roles, with HvNTR2 and HvTrxh1 being the predominant isoforms in the aleurone layer.
Functions of Chloroplastic Adenylate Kinases in Arabidopsis
Authors: Lange Peter Robert , Geserick Claudia , Tischendorf Gilbert , Zrenner Rita
Published: December 2007

Adenosine monophosphate kinase (AMK; adenylate kinase) catalyses the reversible formation of ADP by the transfer of one phosphate group from ATP to AMP, thus equilibrating adenylates. The Arabidopsis (Arabidopsis thaliana) genome contains 10 genes with an adenylate/cytidylate kinase signature; seven of these are identified as putative adenylate kinases. Encoded proteins of at least two members of this Arabidopsis adenylate kinase gene family are targeted to plastids. However, when the individual genes are disrupted, the phenotypes of both mutants are strikingly different. Although absence of AMK2 causes only 30% reduction of total adenylate kinase activity in leaves, there is loss of chloroplast integrity leading to small, pale-looking plantlets from embryo to seedling development. In contrast, no phenotype for disruption of the second plastid adenylate kinase was found. From this analysis, we conclude that AMK2 is the major activity for equilibration of adenylates and de novo synthesis of ADP in the plastid stroma.
Silica-Based Thin Films by PE-CVD: An XPS Characterization
Authors: Barreca Davide , Gasparotto Alberto , Maccato Chiara , Maragno Cinzia , Tondello Eugenio
Published: December 2007

Hybrid silica-based (SiOxCyHz) thin films were deposited via plasma enhanced-chemical vapor deposition (PE-CVD) on SiO2, Si(100) and Cu substrates starting from tetramethoxysilane (TMOS) as precursor compound. Depositions were performed from Ar/TMOS plasmas in the absence of oxidizing gases at temperatures as low as 60 °C. In particular, the dependence of the system composition, structure, morphology and optical properties on the applied rf-power (from 20 to 80 W) was investigated in detail. This work is devoted to the XPS (x-ray photoelectron spectroscopy) characterization of a representative SiOxCyHz thin film supported on Si(l00) and synthesized at 80 W, with particular regard to the origin of the organic residuals incorporated in the obtained hybrid material.
Polyolefin Homopolymer and Copolymer Spectra by ToF-SIMS
Authors: Abmayr David W.
Published: December 2007

Time-of-flight secondary ion mass spectrometry spectra (positive ion) of 25 different polymers are presented. The presented spectra are primarily semicrystalline thermoplastic polyolefins and elastomeric polyolefins, but include several other commonly used homopolymers and copolymers. The spectra data set includes: high-density polyethylene, polypropylene, ethylene-propylene rubber, poly 1-butene, ethylene-butene copolymer, poly 4-methyl 1-pentene, poly 1-hexene, ethylene-vinyl acetate copolymer, polyisobutylene, butyl rubber, brominated polyisobutylene 4-methylstyrene rubber, ethylene-methyl acrylate copolymer, polybutadiene, polyisoprene, natural rubber, polychloroprene, polystyrene, styrene-butadiene rubber, and poly 4-methylstyrene, polyethylene glycol, polyethylene terephthalate, and polytetrafluoroethylene. These spectra can be used for the comparitive identification of unknown polymers.