bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatitis C virus‐specific CD8+ T cell frequencies are associated with the responses of pegylated interferon‐α and ribavirin combination therapy in patients with chronic hepatitis C virus infection
Authors: Tatsumi Tomohide , Takehara Tetsuo , Miyagi Takuya , Nakazuru Shoichi , Mita Eiji , Kanto Tatsuya , Hiramatsu Naoki , Hayashi Norio
Published: October 2010

Aim:  Hepatitis C virus (HCV)‐specific cytotoxic T lymphocytes (CTLs) play critical roles in elimination of the HCV‐infected hepatocytes. However, the mechanism of HCV elimination by pegylated interferon‐α (peg‐IFNα) plus ribavirin is not fully understood. We examined HCV‐specific CTL responses during this combination therapy.

Methods:  CD8+ T cells were isolated from 16 HCV infected patients treated by this combination therapy and were subjected to IFN‐γ enzyme‐linked immunospot (ELISPOT) assay.

Results:  The numbers of IFN‐γ spots against HCV Core or NS3 protein‐derived peptides in HCV patients before treatment were similar to those in healthy donors, and those in HCV patients significantly increased 4 weeks after the initiation of combination therapy. All HCV Core or NS3 proteins‐derived peptides specific CD8+ T cells responses in pre‐treated patients were not associated with ALT levels and HCV viral loads of HCV patients before treatment. And those in pre‐treated patients were similar between sustained virologic responder (SVR) patients and non‐SVR patients. Significant increase of HCV Core or NS3 proteins‐derived peptides specific CD8+ T cells responses between before and 4 weeks after this combination therapy were observed in SVR patients, but not in non‐SVR patients.

Conclusions:  These results demonstrated that significant increase of HCV‐specific CD8+ T cells at 4 weeks after the initiation of IFN treatment might be associated with the elimination of HCV. Our findings suggest that the reactivity against HCV Core and NS3 proteins‐derived peptides might be useful in predicting the clinical outcome of the combination therapy of peg‐IFNα and ribavirin.
Unique structure of the M loop region of β1‐tubulin may contribute to size variability of platelets in the family Felidae
Authors: Boudreaux Mary K. , Osborne Christina D. , Herre Adam C. , Rivera Elliot Ramos , Spangler Elizabeth A.
Published: October 2010

Background:Platelet size is relatively uniform in mammals except for domestic cats. Uniform platelet production by megakaryocytes can be disrupted if microtubule assembly or dynamics is impaired. Mutations in the gene encoding β1‐tubulin have been documented in dogs and people, and the resulting microtubule effects have been associated with production of large platelets.

Objectives:The objectives of this study were to evaluate morphology of platelets on feline blood smears, determine the gene sequences encoding β1‐tubulin in members of the family Felidae, and compare the findings with those in other mammalian species to determine whether predicted structural differences in β1‐tubulin that might affect microtubule stability or assembly were present.

Methods:At least 100 platelets/smear on blood smears from 15 domestic cats and 88 big cats were evaluated to assess platelet size variability. Platelet‐derived cDNA obtained from a domestic cat and genomic DNA isolated from blood samples of domestic cats and other members of the family Felidae were analyzed by PCR using primers specific for β1‐tubulin. Gene sequences obtained were compared with those of other common mammals.

Results:Two differences in gene sequence were found in a highly conserved region encoding the M loop of β1‐tubulin in members of the family Felidae compared with sequences from other species. Platelet size variation was present in big cats and domestic cats. In addition, a rare amino acid change was documented in the C‐terminal region encoding the H11 helix in domestic cats.

Conclusion:Members of the family Felidae have an altered M loop region in β1‐tubulin compared with other mammals. This variation may contribute to the observed platelet size variability.
CFD Based Ash Deposition Prediction in a BFBC Firing Mixtures of Peat and Forest Residue
Authors: Lundmark Dan , Mueller Christian , Backman Rainer , Zevenhoven Maria , Skrifvars Bengt-Johan , Hupa Mikko
Published: October 2010

Combustion of biofuel mixtures for heat and power production is often combined with ash related operational problems. Their reliable prediction is therefore essential in order to improve boiler performance and availability. In this work, an ash behavior prediction tool based on computational fluid dynamics and advanced fuel analysis is used to predict the ash deposition tendencies in a 295 MWth bubbling fluidized bed boiler fired with mixtures of peat and forest residue. The work focuses especially on two new modeling approaches for fuel distribution and bubbling bed. The overall combustion and deposition prediction results agree well with conditions in a real boiler.
Modeling and Experimental Study of Newtonian Fluid Flow in Annulus
Authors: Sorgun Mehmet , Ozbayoglu M. Evren , Aydin Ismail
Published: October 2010

A major concern in drilling operations is the proper determination of frictional pressure loss in order to select a mud pump and avoid any serious problems. In this study, a mechanistic model is proposed for predicting the frictional pressure losses of light drilling fluid, which can be used for concentric annuli. The experimental data that were available in the literature and conducted at the Middle East Technical University-Petroleum Engineering (METU-PETE) flow loop as well as computational fluid dynamics (CFD) software are used to verify the results from the proposed mechanistic model. The results showed that the proposed model can estimate frictional pressure losses within a ±10% error interval when compared with the experimental data. Additionally, the effect of the pipe eccentricity on frictional pressure loss and tangential velocity using CFD for laminar and turbulent flow is also examined. It has been observed that pipe eccentricity drastically increases the tangential velocity inside the annulus; especially, the flow regime is turbulent and frictional pressure loss decreases as the pipe eccentricity increases.
Block Copolymer Self-Assembly–Directed Single-Crystal Homo- and Heteroepitaxial Nanostructures
Authors: Arora Hitesh , Du Phong , Tan Kwan W. , Hyun Jerome K. , Grazul John , Xin Huolin L. , Muller David A. , Thompson Michael O. , Wiesner Ulrich
Published: October 2010

Polymer Templating for Metals

Polymer templating has been used to fabricate a wide range of ordered materials, both due to the ability to pattern the polymers easily over a large area and their facile removal. However, the process is somewhat limited to the incorporation of materials that will flow easily into the templated areas.

Arora
et al.

(p.
214
) show that current techniques can be extended to the patterning of metals, through guided epitaxial growth. An excimer laser was used to control the flow of material into patterned templates formed from block copolymers.
The Dynamics of the Onset of Frictional Slip
Authors: Ben-David Oded , Cohen Gil , Fineberg Jay
Published: October 2010

Slip Sliding Away

The initiation of frictional motion, or slip, along ideal surfaces typically behaves as predicted by rupture models. When stress heterogeneities—similar to irregularities in fault zones in Earth's crust—are introduced, rupture propagation speeds are not as well constrained by models. To improve understanding of slip behavior,

Ben-David
et al.

(p.
211
; see the Perspective by

Zapperi

) measured rupture speeds and stress profiles along an extended frictional interface between two polymer blocks. The experiments revealed a slow mode of slip that occurs when the local ratio of shear to normal stress is sufficiently low. The selection and arrest of three distinct modes of rupture depended on the value of the local stress ratio.
Neuroscientists Grapple With Their Field's Big Questions
Authors: Miller Greg
Published: October 2010

Last week, 18 leading neuroscientists gathered to discuss the major open questions in their field and the as-yet-to-be-invented technological advances that could best help solve them.
A Chill in China-Japan Academic Relations
Authors: Normile Dennis
Published: October 2010

A diplomatic tiff stemming from a collision last month between a Chinese trawler and two Japanese coast guard vessels has chilled academic exchange between the two countries.
Grave Disputes
Authors: Lawler Andrew
Published: October 2010

As U.S. legislation requiring the return of Native American remains to tribes turns 20, a new controversy is threatening the tenuous relations between the scientific and Native American communities.
Honoring His Ancestor by Studying His DNA
Authors: Travis John
Published: October 2010

Ernie LaPointe, great-grandson of famed Lakota warrior Sitting Bull, has entered into an unusual partnership with geneticist Eske Willerslev to analyze DNA from a lock of Sitting Bull's hair. LaPointe spoke with
Science
recently; this conversation has been edited for brevity and clarity.
Penetration of Drugs through the Blood-Cerebrospinal Fluid/Blood-Brain Barrier for Treatment of Central Nervous System Infections
Authors: Nau Roland , Sörgel Fritz , Eiffert Helmut
Published: October 2010

The entry of anti-infectives into the central nervous system (CNS) depends on the compartment studied, molecular size, electric charge, lipophilicity, plasma protein binding, affinity to active transport systems at the blood-brain/blood-cerebrospinal fluid (CSF) barrier, and host factors such as meningeal inflammation and CSF flow. Since concentrations in microdialysates and abscesses are not frequently available for humans, this review focuses on drug CSF concentrations. The ideal compound to treat CNS infections is of small molecular size, is moderately lipophilic, has a low level of plasma protein binding, has a volume of distribution of around 1 liter/kg, and is not a strong ligand of an efflux pump at the blood-brain or blood-CSF barrier. When several equally active compounds are available, a drug which comes close to these physicochemical and pharmacokinetic properties should be preferred. Several anti-infectives (e.g., isoniazid, pyrazinamide, linezolid, metronidazole, fluconazole, and some fluoroquinolones) reach a CSF-to-serum ratio of the areas under the curves close to 1.0 and, therefore, are extremely valuable for the treatment of CNS infections. In many cases, however, pharmacokinetics have to be balanced againstin vitroactivity. Direct injection of drugs, which do not readily penetrate into the CNS, into the ventricular or lumbar CSF is indicated when other effective therapeutic options are unavailable.
Melanized Fungi in Human Disease
Authors: Revankar Sanjay G. , Sutton Deanna A.
Published: October 2010

Melanized or dematiaceous fungi are associated with a wide variety of infectious syndromes. Many are soil organisms and are generally distributed worldwide, though certain species appear to have restricted geographic ranges. Though they are uncommon causes of disease, melanized fungi have been increasingly recognized as important pathogens, with most reports occurring in the past 20 years. The spectrum of diseases with which they are associated has also broadened and includes allergic disease, superficial and deep local infections, pneumonia, brain abscess, and disseminated infection. For some infections in immunocompetent individuals, such as allergic fungal sinusitis and brain abscess, they are among the most common etiologic fungi. Melanin is a likely virulence factor for these fungi. Diagnosis relies on careful microscopic and pathological examination, as well as clinical assessment of the patient, as these fungi are often considered contaminants. Therapy varies depending upon the clinical syndrome. Local infection may be cured with excision alone, while systemic disease is often refractory to therapy. Triazoles such as voriconazole, posaconazole, and itraconazole have the most consistentin vitroactivity. Further studies are needed to better understand the pathogenesis and optimal treatment of these uncommon infections.
Immunization with Truncated Recombinant Protein SpaC ofErysipelothrix rhusiopathiaeStrain 715 Serovar 18 Confers Protective Immunity against Challenge with Various Serovars
Authors: To Ho , Someno Shuichi , Nagai Shinya , Koyama Tomohiro , Nagano Tetsuji
Published: October 2010

Previously, we showed that surface protective antigen (Spa) proteins ofErysipelothrix rhusiopathiaecan be classified into three molecular species—SpaA, SpaB, and SpaC—and that SpaC is the most broadly cross-protective antigen among the three Spa proteins. In this study, we examined the ability of the α-helical domain, which comprises the N-terminal half of SpaC, to elicit cross-protective immunity in mice and pigs. Mice actively immunized with the full-length protein (rSpaC664) or the α-helical domain (rSpaC427), but not the C-terminal domain (rSpaC253), were protected against challenge withE. rhusiopathiaeserovars 1a, 2, 6, 19, and 18 expressing heterologous (SpaA or SpaB) and homologous (SpaC) Spas. The α-helical domain seemed to provide better protection than rSpaC664, although the differences did not reach statistical significance. Similarly, mice passively immunized with rabbit anti-rSpaC664 or anti-rSpaC427 sera, but not anti-rSpaC253 serum, were protected from challenge with various serovars. Pigs immunized with SpaC427 also developed specific antibodies against Spa proteins and were protected from challenge with the highly virulent heterologousE. rhusiopathiaestrain Fujisawa (serovar 1a). Taken together, these results demonstrate for the first time the striking protective efficacy of the α-helical domain-mediated immunization in both mice and pigs, thereby highlighting its utility as the most promising candidate for the development of a safe and effective vaccine against erysipelas.
Seroprevalence of Pertussis among Danish Patients with Cough of Unknown Etiology
Authors: Dalby Tine , Harboe Zitta B. , Krogfelt Karen Angeliki
Published: October 2010

The common perception that pertussis is only a childhood disease is not correct. Vaccination or infection with
Bordetella pertussis
provides only short-lived protection against pertussis—and the majority of the population is consequently at risk of contracting pertussis. We evaluated the seroprevalence of pertussis antibodies (IgG against pertussis toxin) in serum samples from 265 Danish patients, aged 8 years and older, with coughs of unknown etiology. Depending on the cutoff chosen, we found that 2.6% to 10.9% of these patients were seropositive for pertussis. Of 178 patients with a reported duration of cough between 2 weeks and 3 months, 3.4% to 12.4% were seropositive for pertussis, indicating recent infection. Our study indicates that
B. pertussis
infection may be underdiagnosed among older children and adults with coughs in Denmark.
Contributions of Conventional and Heavy-Chain IgG to Immunity in Fetal, Neonatal, and Adult Alpacas
Authors: Daley-Bauer L. P. , Purdy S. R. , Smith M. C. , Gagliardo L. F. , Davis W. C. , Appleton J. A.
Published: October 2010

In addition to conventional immunoglobulins, camelids produce antibodies that do not incorporate light chains into their structures. These so-called heavy-chain (HC) antibodies have incited great interest in the biomedical community, as they have considerable potential for biotechnological and therapeutic application. Recently, we have begun to elucidate the immunological functions of HC antibodies, yet little is known about their significance in maternal immunity or about the B lymphocytes that produce them. This study describes the application of isotype-specific reagents toward physiological assessments of camelid IgGs and the B cells that produce them. We document the specificities of monoclonal antibodies that distinguish two conventional IgG1 isotypes and two HC IgG3 variants produced by alpacas. Next, we report that the relative concentrations of five isotypes are similar in serum, milk, and colostrum; however, following passive transfer, the concentrations of HC IgG2 and IgG3 declined more rapidly than the concentration of conventional IgG1 in the sera of neonates. Finally, we assessed the distribution of B cells of distinct isotypes within lymphoid tissues during fetal and adult life. We detected IgG1, IgG2, and IgG3 in lymphocytes located in lymph node follicles, suggesting that HC B cells affinity mature and/or class switch. One IgG3 isotype was present in B cells located in ileal Peyer's patches, and one conventional IgG1 isotype was detected in splenic marginal zone B cells. Our findings contribute to the growing body of knowledge pertaining to HC antibodies and are compatible with functional specialization among conventional and HC IgGs in the alpaca.
Molecular Cloning of an Immunogenic Protein ofBaylisascaris procyonisand Expression inEscherichia colifor Use in Developing Improved Serodiagnostic Assays
Authors: Dangoudoubiyam Sriveny , Vemulapalli Ramesh , Hancock Kathy , Kazacos Kevin R.
Published: October 2010

Larva migrans caused byBaylisascaris procyonisis an important zoonotic disease. Current serological diagnostic assays for this disease depend on the use of the parasite's larval excretory-secretory (ES) antigens. In order to identify genes encoding ES antigens and to generate recombinant antigens for use in diagnostic assays, construction and immunoscreening of aB. procyonisthird-stage larva cDNA expression library was performed and resulted in identification of a partial-length cDNA clone encoding an ES antigen, designated repeat antigen 1 (RAG1). The full-lengthrag1cDNA contained a 753-bp open reading frame that encoded a protein of 250 amino acids with 12 tandem repeats of a 12-amino-acid long sequence. Therag1genomic DNA revealed a single intron of 837 bp that separated the 753-bp coding sequence into two exons delimited by canonical splice sites. No nucleotide or amino acid sequences present in the GenBank databases had significant similarity with those of RAG1. We have cloned, expressed, and purified the recombinant RAG1 (rRAG1) and analyzed its diagnostic potential by enzyme-linked immunosorbent assay. Anti-Baylisascarisspecies-specific rabbit serum showed strong reactivity to rRAG1, while only minimal to no reactivity was observed with sera against the related ascaridsToxocara canisandAscaris suum, strongly suggesting the specificity of rRAG1. On the basis of these results, the identified RAG1 appears to be a promising diagnostic antigen for the development of serological assays for specific detection ofB. procyonislarva migrans.
Improved Detection of
Bartonella
DNA in Mammalian Hosts and Arthropod Vectors by Real-Time PCR Using the NADH Dehydrogenase Gamma Subunit (
nuoG
)
Authors: Colborn James M. , Kosoy Michael Y. , Motin Vladimir L. , Telepnev Maxim V. , Valbuena Gustavo , Myint Khin S. , Fofanov Yuri , Putonti Catherine , Feng Chen , Peruski Leonard
Published: October 2010

We used a whole-genome scanning technique to identify the NADH dehydrogenase gamma subunit (
nuoG
) primer set that is sensitive and specific enough to detect a diverse number of
Bartonella
species in a wide range of environmental samples yet maintains minimal cross-reactivity to mammalian host and arthropod vector organisms.
Incidence of and Risk Factors for Community-Associated Methicillin-Resistant
Staphylococcus aureus
Acquired Infection or Colonization in Intensive-Care-Unit Patients
Authors: Wang Jann-Tay , Liao Chun-Hsing , Fang Chi-Tai , Chie Wei-Chu , Lai Mei-Shu , Lauderdale Tsai-Ling , Chang Shan-Chwen
Published: October 2010

The incidence of and risk factors for acquiring community-associated methicillin-resistant
Staphylococcus aureus
(CA-MRSA) among patients staying in intensive care units (ICUs) remain unclear. We enrolled patients staying in two ICUs at the Far Eastern Memorial Hospital during the period of 1 September 2008 to 30 September 2009 to clarify this issue. Surveillance cultures for MRSA were taken from nostril, sputum or throat, axillae, and the inguinal area in all enrolled patients upon admission to the ICU, every 3 days thereafter, and on the day of discharge from the ICU. For each MRSA isolate, we performed multilocus sequence typing, identified the type of staphylococcal cassette chromosome
mec
, detected the presence of the Panton-Valentine leukocidin gene, and conducted drug susceptibility tests. Among the 1,906 patients who were screened, 203 patients were carriers of MRSA before their admission to the ICU; 81 patients acquired MRSA during their stay in the ICU, including 31 who acquired CA-MRSA. The incidence rates of newly acquired MRSA and CA-MRSA during the ICU stay were 7.9 and 3.0 per 1,000 patient-days, respectively. Prior usage of antipseudomonal penicillins and antifungals and the presence of a nasogastric tube were found to be independent risk factors for acquiring CA-MRSA during the ICU stay when data for CA-MRSA carriers and patients without carriage of MRSA were compared (
P
= 0.0035, 0.0330, and 0.0262, respectively). Prior usage of carbapenems was found to be a protective factor against acquiring CA-MRSA when data for patients with CA-MRSA and those with health care-associated MRSA acquired during ICU stay were compared (
P
= 0.0240).
Susceptibility Profiles, Molecular Epidemiology, and Detection of KPC-Producing
Escherichia coli
Isolates from the New York City Vicinity
Authors: Landman David , Urban Carl , Bäcker Martin , Kelly Paul , Shah Neha , Babu Elizabeth , Bratu Simona , Quale John
Published: October 2010

The detection of
Enterobacteriaceae
carrying the carbapenemase KPC has been problematic. Thirty isolates of KPC-possessing
Escherichia coli
were gathered from hospitals in New York City and Connecticut. The imipenem, meropenem, doripenem, and ertapenem MIC
50
values were 4, 2, 1, and 4 μg/ml, respectively. Over half of the isolates belonged to a single ribotype. Using an ertapenem breakpoint of 0.25 μg/ml would efficiently detect these isolates.
HIV-1 Continues To Replicate and Evolve in Patients with Natural Control of HIV Infection
Authors: Mens Helene , Kearney Mary , Wiegand Ann , Shao Wei , Schønning Kristian , Gerstoft Jan , Obel Niels , Maldarelli Frank , Mellors John W. , Benfield Thomas , Coffin John M.
Published: October 2010

Elucidating mechanisms leading to the natural control of HIV-1 infection is of great importance for vaccine design and for understanding viral pathogenesis. Rare HIV-1-infected individuals, termed HIV-1 controllers, have plasma HIV-1 RNA levels below the limit of detection by standard clinical assays (<50 to 75 copies/ml) without antiretroviral therapy. Although several recent studies have documented persistent low-grade viremia in HIV-1 controllers at a level not significantly different from that in HIV-1-infected individuals undergoing treatment with combination antiretroviral therapy (cART), it is unclear if plasma viruses are undergoing full cycles of replication
in vivo
or if the infection of new cells is completely blocked by host immune mechanisms. We studied a cohort of 21 HIV-1 controllers with a median level of viremia below 1 copy/ml, followed for a median of 11 years. Less than half of the cohort carried known protective HLA types (B*57/27). By isolating HIV-1 RNA from large volumes of plasma, we amplified single genome sequences of both
pro-rt
and
env
longitudinally. This study is the first to document that HIV-1
pro-rt
and
env
evolve in this patient group, albeit at rates somewhat lower than in HIV-1 noncontrollers, in HLA B*57/27-positive, as well as HLA B*57/27-negative, individuals. Viral diversity and adaptive events associated with immune escape were found to be restricted in HIV-1 controllers, suggesting that replication occurs in the face of less overall immune selection.
Glycine 184 in Nonstructural Protein NS1 Determines the Virulence of Influenza A Virus Strain PR8 without Affecting the Host Interferon Response
Authors: Steidle Sabine , Martínez-Sobrido Luis , Mordstein Markus , Lienenklaus Stefan , García-Sastre Adolfo , Stäheli Peter , Kochs Georg
Published: October 2010

The nonstructural protein NS1 of influenza A virus counteracts the interferon (IFN) system and thereby promotes viral replication. NS1 has acquired different mechanisms to limit induction of IFN. It prevents double-stranded RNA (dsRNA) and RIG-I-mediated activation of interferon regulatory factor 3 (IRF3), and it blocks posttranscriptional processing of cellular mRNAs by binding to the cleavage and polyadenylation specificity factor (CPSF). Using a mouse-adapted A/PR/8/34 virus and reverse genetics to introduce specific mutations in NS1 which eliminate one or both functions, we determined the relative contributions of these two activities of NS1 to viral virulence in mice. We found that a functional RNA-binding motif was required for IFN suppression and virulence. Restoration of CPSF binding in the NS1 protein of wild-type A/PR/8/34 virus, which cannot bind CPSF due to mutations in the central binding motif at positions 103 and 106, resulted in enhanced virulence. Surprisingly, if CPSF binding was abolished by substituting glycine for arginine at position 184 in the classical NS1-CPSF binding motif, the mutant virus replicated much more slowly in mice, although the mutated NS1 protein continued to repress the IFN response very efficiently. Our results show that a functional RNA-binding motif is decisive for NS1 of A/PR/8/34 virus to suppress IFN induction. They further demonstrate that in addition to its contribution to CPSF binding, glycine 184 strongly influences viral virulence by an unknown mechanism which does not involve the IFN system.
Multiple Functions of
Rice Dwarf Phytoreovirus
Pns10 in Suppressing Systemic RNA Silencing
Authors: Ren Bo , Guo Yuanyuan , Gao Feng , Zhou Peng , Wu Feng , Meng Zheng , Wei Chunhong , Li Yi
Published: October 2010

RNA silencing is a potent mechanism of antiviral defense response in plants and other organisms. For counterdefense, viruses have evolved a variety of suppressors of RNA silencing (VSRs) that can inhibit distinct steps of a silencing pathway. We previously identified Pns10 encoded by
Rice dwarf phytoreovirus
(RDV) as a VSR, the first of its kind from double-stranded RNA (dsRNA) viruses. In this study we investigated the mechanisms of Pns10 function in suppressing systemic RNA silencing in the widely used
Nicotiana benthamiana
model plant. We report that Pns10 suppresses local and systemic RNA silencing triggered by sense mRNA, enhances viral replication and/or viral RNA stability in inoculated leaves, accelerates the systemic spread of viral infection, and enables viral invasion of shoot apices. Mechanistically, Pns10 interferes with the perception of silencing signals in recipient tissues, binds double-stranded small interfering RNA (siRNAs) with two-nucleotide 3′ overhangs, and causes the downregulated expression of RDR6. These results significantly deepen our mechanistic understanding of the VSR functions encoded by a dsRNA virus and contribute additional evidence that binding siRNAs and interfering with RDR6 expression are broad mechanisms of VSR functions encoded by diverse groups of viruses.
Human T-Lymphotropic Virus Type 1 Tax Protein Complexes with P-TEFb and Competes for Brd4 and 7SK snRNP/HEXIM1 Binding
Authors: Cho Won-Kyung , Jang Moon Kyoo , Huang Keven , Pise-Masison Cynthia A. , Brady John N.
Published: October 2010

Positive transcription elongation factor b (P-TEFb) plays an important role in stimulating RNA polymerase II elongation for viral and cellular gene expression. P-TEFb is found in cells in either an active, low-molecular-weight (LMW) form or an inactive, high-molecular-weight (HMW) form. We report here that human T-lymphotropic virus type 1 (HTLV-1) Tax interacts with the cyclin T1 subunit of P-TEFb, forming a distinct Tax/P-TEFb LMW complex. We demonstrate that Tax can play a role in regulating the amount of HMW complex present in the cell by decreasing the binding of 7SK snRNP/HEXIM1 to P-TEFb. This is seen both
in vitro
using purified Tax protein and
in vivo
in cells transduced with Tax expression constructs. Further, we find that a peptide of cyclin T1 spanning the Tax binding domain inhibits the ability of Tax to disrupt HMW P-TEFb complexes. These results suggest that the direct interaction of Tax with cyclin T1 can dissociate P-TEFb from the P-TEFb/7SK snRNP/HEXIM1 complex for activation of the viral long terminal repeat (LTR). We also show that Tax competes with Brd4 for P-TEFb binding. Chromatin immunoprecipitation (ChIP) assays demonstrated that Brd4 and P-TEFb are associated with the basal HTLV-1 LTR, while Tax and P-TEFb are associated with the activated template. Furthermore, the knockdown of Brd4 by small interfering RNA (siRNA) activates the HTLV-1 LTR promoter, which results in an increase in viral expression and production. Our studies have identified Tax as a regulator of P-TEFb that is capable of affecting the balance between its association with the large inactive complex and the small active complex.
Tetrameric Ring Formation of Epstein-Barr Virus Polymerase Processivity Factor Is Crucial for Viral Replication
Authors: Nakayama Sanae , Murata Takayuki , Yasui Yoshihiro , Murayama Kazutaka , Isomura Hiroki , Kanda Teru , Tsurumi Tatsuya
Published: October 2010

The Epstein-Barr virus BMRF1 DNA polymerase processivity factor, which is essential for viral genome replication, exists mainly as a C-shaped head-to-head homodimer but partly forms a ring-shaped tetramer through tail-to-tail association. Based on its molecular structure, several BMRF1 mutant viruses were constructed to examine their influence on viral replication. The R256E virus, which has a severely impaired capacity for DNA binding and polymerase processivity, failed to form replication compartments, resulting in interference of viral replication, while the C95E mutation, which impairs head-to-head contact
in vitro
, unexpectedly hardly affected the viral replication. Also, surprisingly, replication of the C206E virus, which is expected to have impairment of tail-to-tail contact, was severely restricted, although the mutant protein possesses the same
in vitro
biochemical activities as the wild type. Since the tail-to-tail contact surface is smaller than that of the head-to-head contact area, its contribution to ring formation might be essential for viral replication.
Immature and Transitional B Cells Are Latency Reservoirs for a Gammaherpesvirus
Authors: Coleman Carrie B. , Nealy Michael S. , Tibbetts Scott A.
Published: October 2010

Gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 [HHV-8]), Epstein-Barr virus (EBV), and murine gammaherpesvirus 68 (MHV68; also known as gammaherpesvirus 68 [γHV68] or murine herpesvirus 4 [MuHV-4]), establish lifelong latency in the resting memory B cell compartment. However, little is known about how this reservoir of infected mature B cells is maintained for the life of the host. In the context of a normal immune system, the mature B cell pool is naturally maintained by the renewable populations of developing B cells that arise from hematopoiesis. Thus, recurrent infection of these developing B cell populations could allow the virus continual access to the B cell lineage and, subsequent to differentiation, the memory B cell compartment. To begin to address this hypothesis, we examined whether MHV68 establishes latency in developing B cells during a normal course of infection. In work described here, we demonstrate the presence of viral genome in bone marrow pro-pre-B cells and immature B cells during early latency and immature B cells during long-term latency. Further, we show that transitional B cells in the spleen are latently infected and express the latency-associated nuclear antigen (LANA) throughout chronic infection. Because developing B cells normally exhibit a short life span and a high rate of turnover, these findings suggest a model in which gammaherpesviruses may gain access to the mature B cell compartment by recurrent seeding of developing B cells.
Interactions Between Nuclei and the Cytoskeleton Are Mediated by SUN-KASH Nuclear-Envelope Bridges
Authors: Starr Daniel A. , Fridolfsson Heidi N.
Published: October 2010

The nuclear envelope links the cytoskeleton to structural components of the nucleus. It functions to coordinate nuclear migration and anchorage, organize chromatin, and aid meiotic chromosome pairing. Forces generated by the cytoskeleton are transferred across the nuclear envelope to the nuclear lamina through a nuclear-envelope bridge consisting of SUN (Sad1 and UNC-84) and KASH (Klarsicht, ANC-1 and Syne/Nesprin homology) proteins. Some KASH-SUN combinations connect microtubules, centrosomes, actin filaments, or intermediate filaments to the surface of the nucleus. Other combinations are used in cell cycle control, nuclear import, or apoptosis. Interactions between the cytoskeleton and the nucleus also affect global cytoskeleton organization. SUN and KASH proteins were identified through genetic screens for mispositioned nuclei in model organisms. Knockouts of SUN or KASH proteins disrupt neurological and muscular development in mice. Defects in SUN and KASH proteins have been linked to human diseases including muscular dystrophy, ataxia, progeria, lissencephaly, and cancer.
A New Wave of Cellular Imaging
Authors: Toomre Derek , Bewersdorf Joerg
Published: October 2010

Fluorescence imaging methods that push or break the diffraction limit of resolution (approximately 200 nm) have grown explosively. These super-resolution nanoscopy techniques include: stimulated emission depletion (STED), Pointillism microscopy [(fluorescence) photoactivation localization microscopy/stochastic optical reconstruction microscopy, or (F)PALM/STORM], structured illumination, total internal reflection fluorescence microscopy (TIRFM), and those that combine multiple modalities. Each affords unique strengths in lateral and axial resolution, speed, sensitivity, and fluorophore compatibility. We examine the optical principles and design of these new instruments and their ability to see more detail with greater sensitivity—down to single molecules with tens of nanometers resolution. Nanoscopes have revealed transient intermediate states of organelles and molecules in living cells and have led to new discoveries but also biological controversies. We highlight common unifying principles behind nanoscopy such as the conversion of a subset of probes between states (ground or excited) and the use of scanning (ordered or stochastic). We emphasize major advances, biological applications, and promising new developments.
Novel Research Horizons for Presenilins and γ-Secretases in Cell Biology and Disease
Authors: De Strooper Bart , Annaert Wim
Published: October 2010

Presenilins are the catalytic subunits of larger tetrameric γ-secretase complexes. The degradome of these aspartyl proteases consists of at least 60 different substrates. γ-Secretase is key to regulated intramembrane proteolysis, releasing protein fragments that potentially transduce signals at both sides of the cell membrane. Characteristic for this novel form of cellular signaling is its irreversible nature, providing direction to biological processes. We discuss recent insights in structure-function and assembly of the γ-secretase complexes and emerging insights in the regulation of the activity of these enzymes. This novel knowledge will help to develop better drugs for Alzheimer's disease and cancer. We critically evaluate literature that proposes presenilin functions outside of the γ-secretase complex.
Ubiquitination in Postsynaptic Function and Plasticity
Authors: Mabb Angela M. , Ehlers Michael D.
Published: October 2010

Neurons are highly specialized cells whose connectivity at synapses subserves rapid information transfer in the brain. Proper information processing, learning, and memory storage in the brain requires continuous remodeling of synaptic networks. Such remodeling includes synapse formation, elimination, synaptic protein turnover, and changes in synaptic transmission. An emergent mechanism for regulating synapse function is posttranslational modification through the ubiquitin pathway at the postsynaptic membrane. Here, we discuss recent findings implicating ubiquitination and protein degradation in postsynaptic function and plasticity. We describe postsynaptic ubiquitination pathways and their role in brain development, neuronal physiology, and brain disorders.
Cell Motility and Mechanics in Three-Dimensional Collagen Matrices
Authors: Grinnell Frederick , Petroll W. Matthew
Published: October 2010

Fibrous connective tissues provide mechanical support and frameworks for other tissues of the body and play an integral role in normal tissue physiology and pathology. Three-dimensional collagen matrices exhibit mechanical and structural features that resemble fibrous connective tissue and have become an important model system to study cell behavior in a tissue-like environment. This review focuses on motile and mechanical interactions between cells—especially fibroblasts—and collagen matrices. We describe several matrix contraction models, the interactions between fibroblasts and collagen fibrils at global and subcellular levels, unique features of mechanical feedback between cells and the matrix, and the impact of the cell-matrix tension state on cell morphology and mechanical behavior. We develop a conceptual framework to explain the balance between cell migration and collagen translocation including the concept of promigratory and procontractile growth factor environments. Finally, we review the significance of these concepts for the physiology of wound repair.
The Diverse Functions of Oxysterol-Binding Proteins
Authors: Raychaudhuri Sumana , Prinz William A.
Published: October 2010

Oxysterol-binding protein (OSBP)-related proteins (ORPs) are lipid-binding proteins that are conserved from yeast to humans. They are implicated in many cellular processes including signaling, vesicular trafficking, lipid metabolism, and nonvesicular sterol transport. All ORPs contain an OSBP-related domain (ORD) that has a hydrophobic pocket that binds a single sterol. ORDs also contain additional membrane-binding surfaces, some of which bind phosphoinositides and may regulate sterol binding. Studies in yeast suggest that ORPs function as sterol transporters, perhaps in regions where organelle membranes are closely apposed. Yeast ORPs also participate in vesicular trafficking, although their role is unclear. In mammalian cells, some ORPs function as sterol sensors that regulate the assembly of protein complexes in response to changes in cholesterol levels. This review will summarize recent advances in our understanding of how ORPs bind lipids and membranes and how they function in diverse cellular processes.
Thyroid hormone inhibits ERK phosphorylation in pressure overload-induced hypertrophied mouse hearts through a receptor-mediated mechanism
Authors: Suarez Jorge , Scott Brian T. , Suarez-Ramirez Jorge A. , Chavira Citlalic V. , Dillmann Wolfgang H.
Published: October 2010

Pressure overload-induced cardiac hypertrophy results in a pathological type of hypertrophy with activation of signaling cascades like the extracellular signal-regulated kinase (ERK) pathway, which promotes negative cardiac remodeling and decreased contractile function. In contrast, thyroid hormone mediates a physiological type of hypertrophy resulting in enhanced contractile function. In addition, thyroid hormone action is diminished in pressure overload-induced cardiac hypertrophy. We hypothesized that thyroid hormone status modulates ERK activity and that administration of thyroid hormone could alter the activity of this kinase in cardiac hypertrophy induced by pressure overload. ERK is activated by phosphorylation; accordingly, we investigated phosphorylation of ERK in hearts of control, hypothyroid, and hyperthyroid mice. In addition, the effect of T3 treatment on ERK phosphorylation in hypertrophied hearts from transverse aortic-constricted (TAC) mice was investigated. Results showed that phosphorylated ERK (p-ERK) was decreased by 25% in hyperthyroid mice. In contrast, hypothyroid mice presented increased p-ERK by 80%. TAC mice presented a greater than fourfold increase of p-ERK compared with control mice. Interestingly, T3 administration dramatically canceled TAC-induced ERK phosphorylation (36% lower compared with control). Raf-1 is upstream of the ERK pathway. TAC mice presented a 45% increase in phospho-Raf-1 (Ser338). T3 treatment inhibited this effect of pressure overload and further decreased p-Raf-1 (Ser338) by 37%, compared with control. Overexpression of thyroid hormone receptor-α in cultured cardiomyocytes potentiated the inhibitory effect of T3 on ERK phosphorylation. We concluded that thyroid hormone has an inhibitory effect on the Raf-1/ERK pathway. Furthermore, treatment of TAC mice with T3 inhibited Raf-1/ERK pathway by a thyroid hormone receptor-dependent mechanism.
Inhibition of voltage-gated K+ channels in dendritic cells by rapamycin
Authors: Tyan Leonid , Sopjani Mentor , Dërmaku-Sopjani Miribane , Schmid Evi , Yang Wenting , Xuan Nguyen Thi , Shumilina Ekaterina , Lang Florian
Published: October 2010

Rapamycin, an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR), is a widely used immunosuppressive drug. Rapamycin affects the function of dendritic cells (DCs), antigen-presenting cells participating in the initiation of primary immune responses and the establishment of immunological memory. Voltage-gated K+ (Kv) channels are expressed in and impact on the function of DCs. The present study explored whether rapamycin influences Kv channels in DCs. To this end, DCs were isolated from murine bone marrow and ion channel activity was determined by whole cell patch clamp. To more directly analyze an effect of mTOR on Kv channel activity, Kv1.3 and Kv1.5 were expressed in Xenopus oocytes with or without the additional expression of mTOR and voltage-gated currents were determined by dual-electrode voltage clamp. As a result, preincubation with rapamycin (0–50 nM) led to a gradual decline of Kv currents in DCs, reaching statistical significance within 6 h and 50 nM of rapamycin. Rapamycin accelerated Kv channel inactivation. Coexpression of mTOR upregulated Kv1.3 and Kv1.5 currents in Xenopus oocytes. Furthermore, mTOR accelerated Kv1.3 channel activation and slowed down Kv1.3 channel inactivation. In conclusion, mTOR stimulates Kv channels, an effect contributing to the immunomodulating properties of rapamycin in DCs.
Tetrahydrobiopterin does not affect end-organ responsiveness to norepinephrine-mediated vasoconstriction in aged skin
Authors: Lang James A. , Holowatz Lacy A. , Kenney W. Larry
Published: October 2010

We have recently demonstrated that tetrahydrobiopterin (BH4) augments reflex vasoconstriction (VC) in aged skin. Although this appears to occur through its role in norepinephrine (NE) biosynthesis, the extent with which vascular mechanisms are affected are unknown. We hypothesized that localized BH4 supplementation would not affect the VC response to exogenous NE when sympathetic nerves were blocked. Two microdialysis fibers were placed in bretylium tosylate pretreated (presynaptically blocks neurotransmitter release from sympathetic adrenergic nerve terminals; iontophoresis, 200 μA for 20 min) 3-cm2 forearm skin of 10 young (Y) and 10 older (O) subjects for perfusion of 1) Ringer (control) and 2) 5 mM BH4. While local skin temperature was clamped at 34°C, six concentrations of NE (10−12, 10−10, 10−8, 10−6, 10−4, 10−2 M) were infused at each drug-treated site. Cutaneous vascular conductance (CVC) was calculated (CVC = laser Doppler flux/mean arterial pressure) and normalized to baseline (%ΔCVCbase). Despite prejunctional adrenergic blockade, NE-mediated VC was blunted in aged skin at each NE dose (10−12: −12 ± 2 vs. −21 ± 2; 10−10: −15 ± 2 vs. −27 ± 1; 10−8: −22 ± 2 vs. −32 ± 2; 10−6: −27 ± 2 vs. −38 ± 1; 10−4: −52 ± 3 vs. −66 ± 5; 10−2: −62 ± 3 vs. −75 ± 4%ΔCVCbase; P < 0.01), and this response was not affected by pretreatment with BH4 ( P > 0.05). Localized BH4 did not affect end-organ responsiveness to exogenous NE, suggesting that the effects of BH4 on cutaneous VC are primarily isolated to the NE biosynthetic pathway.
Impaired overload-induced hypertrophy is associated with diminished mTOR signaling in insulin-resistant skeletal muscle of the obese Zucker rat
Authors: Katta Anjaiah , Kundla Sudarsanam , Kakarla Sunil K. , Wu Miaozong , Fannin Jacqueline , Paturi Satyanarayana , Liu Hua , Addagarla Hari S. , Blough Eric R.
Published: October 2010

Recent data have suggested that insulin resistance may be associated with a diminished ability of skeletal muscle to undergo hypertrophy (Paturi S, Gutta AK, Kakarla SK, Katta A, Arnold EC, Wu M, Rice KM, Blough ER. J Appl Physiol 108: 7–13, 2010). Here we examine the effects of insulin resistance using the obese Zucker (OZ) rat with increased muscle loading on the regulation of the mammalian target of rapamycin (mTOR) and its downstream signaling intermediates 70-kDa ribosomal protein S6 kinase (p70S6k), ribosomal protein S6 (rpS6), eukaryotic elongation factor 2 (eEF2), and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Compared with that observed in lean Zucker (LZ) rats, the degree of soleus muscle hypertrophy as assessed by changes in muscle wet weight (LZ: 35% vs. OZ: 16%) was significantly less in the OZ rats after 3 wk of muscle overload ( P < 0.05). This diminished growth in the OZ rats was accompanied by significant impairments in the ability of the soleus to undergo phosphorylation of mTOR (Ser2448), p70S6k (Thr389), rpS6 (Ser235/236), and protein kinase B (Akt) (Ser473and Thr308) ( P < 0.05). Taken together, these data suggest that impaired overload-induced hypertrophy in insulin-resistant skeletal muscle may be related to decreases in the ability of the muscle to undergo mTOR-related signaling.
Effect of intracerebroventricular benzamil on cardiovascular and central autonomic responses to DOCA-salt treatment
Authors: Abrams Joanna M. , Engeland William C. , Osborn John W.
Published: October 2010

DOCA-salt treatment increases mean arterial pressure (MAP), while central infusion of benzamil attenuates this effect. The present study used c-Fos immunoreactivity to assess the role of benzamil-sensitive proteins in the brain on neural activity following chronic DOCA-salt treatment. Uninephrectomized rats were instrumented with telemetry transmitters for measurement of MAP and with an intracerebroventricular (ICV) cannula for benzamil administration. Groups included rats receiving DOCA-salt treatment alone, rats receiving DOCA-salt treatment with ICV benzamil, and appropriate controls. At study completion, MAP in vehicle-treated DOCA-salt rats reached 142 ± 4 mmHg. In contrast DOCA-salt rats receiving ICV benzamil had lower MAP (124 ± 3 mmHg). MAP in normotensive controls was 102 ± 3 mmHg. c-Fos immunoreactivity was quantified in the supraoptic nucleus (SON) and across subnuclei of the hypothalamic paraventricular nucleus (PVN), as well as other cardiovascular regulatory sites. Compared with vehicle-treated normotensive controls, c-Fos expression was increased in the SON and all subnuclei of the PVN, but not in other key autonomic nuclei, such as the rostroventrolateral medulla. Moreover, benzamil treatment decreased c-Fos immunoreactivity in the SON and in medial parvocellular and posterior magnocellular neurons of the PVN in DOCA-salt rats but not areas associated with regulation of sympathetic activity. Our results do not support the hypothesis that DOCA-salt increases neuronal activity (as indicated by c-Fos immunoreactivity) of other key regions that regulate sympathetic activity. These results suggest that ICV benzamil attenuates DOCA-salt hypertension by modulation of neuroendocrine-related PVN nuclei rather than inhibition of PVN sympathetic premotor neurons in the PVN and rostroventrolateral medulla.