bluuebunny/crossref_metadata_2025 huggingface dataset:
Population genetics of Phytophthora infestans in Denmark reveals dominantly clonal populations and specific alleles linked to metalaxyl‐M resistance
Authors: Montes M. S. , Nielsen B. J. , Schmidt S. G. , Bødker L. , Kjøller R. , Rosendahl S.
Published: October 2015

Control of the potato late blight pathogen Phytophthora infestans relies heavily on chemicals. The fungicide metalaxyl‐M (Mefenoxam) has played an important role in controlling the disease, but insensitivity to the fungicide in certain isolates is now of major concern. A genetic basis for resistance to metalaxyl suggests the possibility for linking resistance phenotypes to specific population genetic markers, but in order to do this, the population genetic structure and mode of reproduction in a population must first be well described. The dynamics of metalaxyl‐M resistance in the Danish population of P. infestans was characterized over the course of the 2013 growing season, as was the population genetic structure, using simple sequence repeat (SSR) genotypes and single nucleotide polymorphism (SNP)‐based mitochondrial haplotyping of over 80 isolates. Both mating types A1 and A2 were present in most fields, but tests for recombination showed that clonal reproduction dominates in Danish populations. Genotype was not linked to haplotype and no differentiation was observed at the haplotype level, but rather between fields. Resistance phenotypes were linked to specific SSR alleles, demonstrating the potential for a more precise SNP‐based marker system for predicting resistance to metalaxyl‐M.
The effect of training in mindfulness and affect consciousness on the therapeutic environment for patients with psychoses: an explorative intervention study
Authors: Eliassen Birgit K. , Sørlie Tore , Sexton Joseph , Høifødt Tordis S.
Published: October 2015

Rationale

The ward atmosphere and the relationships between patients and staff contribute to the improvement in symptoms and psychiatric patient functioning and satisfaction. The quality of the therapeutic relationship is connected to the degree of self‐reflection and self‐awareness by the staff.

Objective

To examine the effect of two different training programmes for healthcare professionals on the ward atmosphere in wards for psychotic patients.

Design and methods

Exploratory intervention study with two eight‐week interventions of mindfulness training and affect‐consciousness training. The outcome was measured on mindfulness and on relevant subscales of the Ward Atmosphere Scale. Data were collected by means of questionnaires and analysed using paired samples t‐tests and repeated measures anova.

Results

Two groups professionals (n = 27 & n = 23) participated in the study. Improvements indicated that both interventions had a positive impact on the ward atmosphere.

Study limitations

The exploratory design and the small samples size make definitive conclusions difficult. Patient assessment on the ward atmosphere scale would have strengthened the validity of the results.

Conclusion

The ward atmosphere in two different wards for psychotic patients improved after staff training in mindfulness or affect consciousness. There were differences in change profiles for the groups in accordance with the differences in the two interventions.

Practice implications

We recommend awareness on the quality of the ward atmosphere in wards for psychotic patients. Both training in mindfulness and affect consciousness can support healthcare professionals in their awareness.
Why Do Patients with COPD Decline Rehabilitation
Authors: Mathar Helle , Fastholm Pernille , Hansen Ida Rode , Larsen Niels Sandholm
Published: October 2015

Aim

This paper aimed to suggest possible answers to the question: Why do patients with COPD decline pulmonary rehabilitation (PR)?

Method

The study is a metasynthesis inspired by Noblit of the existing qualitative research on the area. The data were collected during 2014. Six studies were found through a systematic literature search in relevant databases. In these six studies, 65 persons were identified as decliners of PR. Four themes were identified from these studies.

Results

The themes identified were as follows: the referral process, transport problems, perception of health and other obligations or priorities. The problems with the referral of patients relate to different areas: the referring health professional's conviction and commitment, and the patients' understanding of the referral. It seems that various transport problems cause decline, for example long distance to the PR centre or the expenses of getting back and forth. Perceptions of health cause decline. Decliners feel too sick to join PR or do not identify themselves as a sick person, and do not want undertake the ‘patient role’. Other obligations or priorities such as work, family obligations and vacations are prioritised on behalf of PR causing decline.

Conclusion

The studies included show patients' rational accounts and reflections on declining PR. The included studies tend to describe accounts for deselection of PR in relation to the preferences and beliefs of the patients rather than including the social and economic variables framing the behaviour and choices of the patients.
Facilitators and barriers for electronic social support
Authors: Yli‐Uotila Tiina , Kaunonen Marja , Pylkkänen Liisa , Suominen Tarja
Published: October 2015

Background

Nonprofit cancer societies play an important role in providing social support for patients with cancer through nonprofit electronic counselling services (ECS) provided by counselling nurses (CNs) with experience in oncology nursing. To date, there exist only few studies addressing the facilitators and barriers for social support of patients with cancer as reported by CNs.

Objective

To describe the facilitators and barriers for electronic social support of patients with cancer received from the ECS in the nonprofit cancer societies as reported by CNs.

Methods

Qualitative design with three group interviews was conducted with 10 CNs in three nonprofit cancer societies in southern and western parts of Finland. Interviews were recorded, transcribed verbatim and content analysed inductively.

Findings

The facilitators were promotion of the access to ECS, functioning structures of ECS, utilisation of the strengths of an individual CN in ECS, promotion of the life management of patients, patient‐centeredness as a basis of ECS and reliability of ECS. The barriers for electronic social support were the unmet paths between ECS and patients, nonfunctioning structures of ECS, inadequacy of mutual communication and lack of shared viewpoints between CNs and patients.

Conclusions

Facilitators and barriers for electronic social support of patients with cancer were related to organisation, individuals and counselling process. The counselling work in ECS as its best promotes the life management of patients with cancer but, alternatively, can lead to conflicts in communication and therefore be a barrier for electronic social support.

Implications for practice

To make the nonprofit ECS better known, the cooperation with hospitals is needed to enable social support for patients. To improve communication between CNs and patients, continuous communications skills training and functional working environments are needed.
Allelic polymorphism of KIR2DL2/2DL3 in a southern Chinese population
Authors: Zhen J. , He L. , Xu Y. , Zhao J. , Yu Q. , Zou H. , Sun G. , Deng Z.
Published: October 2015

KIR2DL2 and KIR2DL3 segregate as alleles of the same killer cell immunoglobulin‐like receptor (KIR) gene locus. They have been associated with viral infectious diseases and certain cancers and their allelic information may help to better comprehend mechanisms. The allelic polymorphism of KIR2DL2/2DL3 has been shown to influence their binding specificity and affinity to the HLA‐C1 ligands. The present study aims to investigate the distribution of the allelic polymorphism of KIR2DL2/2DL3 in a southern Chinese population using sequence‐specific primer polymerase chain reaction (PCR‐SSP) and PCR‐sequence‐based typing (SBT) at the entire coding sequence. Of the 306 tested individuals, 1.96% were positive for KIR2DL2 only, 78.10% for KIR2DL3 only, and 19.93% for both KIR2DL2 and 2DL3. KIR2DL3 showed a high degree of diversity in the study population with 15 alleles detected including 8 novel ones. The predominant 2DL3 allele in the study population is 2DL3*00101 (92.81%) followed by 2DL3*00201 (24.18%), 2DL3*023 (4.25%), and 2DL3*00109 (1.31%). The remaining 11 2DL3 alleles all had a frequency below 1%. Three detected 2DL2 alleles were 2DL2*00301 (18.95%), 2DL2*00101 (3.59%), and the novel 2DL2*013 (0.33%). These results provide further insight into the KIR gene diversity in Southern Chinese and may help to better understand the role played by KIR genes in associated diseases.
The changing importance of key factors associated with anaemia in 6‐ to 59‐month‐old children in a sub‐Saharan African setting where malaria is on the decline: analysis of the Rwanda Demographic and Health Survey 2010
Authors: Nkulikiyinka Richard , Binagwaho Agnes , Palmer Katie
Published: October 2015

Objective

To estimate the relative contribution of malaria and other potential determinants to current anaemia prevalence in Rwanda.

Methods

The database for this study was the Rwanda Demographic and Health Survey 2010. Haemoglobin and malaria test results, and additional exposures ascertained through mothers’ interviews, were analysed for all eligible children age 6–59 months (n = 4068), in addition to diet data available for the youngest under 5‐year‐old per household. We examined anaemia‐exposure associations through forward logistic regression, first for the overall population (n = 3685), and second, for the subpopulation with diet data (n = 1934).

Results

In the overall study population, malaria was strongly associated with anaemia (OR = 6.83, 95% CI: 2.90–16.05), but population impact was modest (population‐attributable fraction = 2.5%). Factors associated with lower odds of anaemia were recent de‐worming medication (six months; OR = 0.60, 95% CI: 0.49–0.74), female sex (OR = 0.76, 95% CI: 0.66–0.87), increasing age, residence in North Province and educated mother. Being underweight and recent fever (two weeks) were associated with higher odds. In the subpopulation with diet data, odds were lower with consumption of vitamin A‐rich foods (OR = 0.66, 95% CI: 0.50–0.88); and higher in households with many young children.

Conclusions

Malaria remains a strong determinant of anaemia for the individual child: transmission control efforts must be maintained. At population level, to further reduce anaemia prevalence, promoting regular vitamin A intake from natural sources and reducing intestinal helminths burden appear the most promising strategies to explore; exploring potential hitherto unidentified sex‐linked factors is warranted.
Identification of immunodominant antigens for the laboratory diagnosis of toxocariasis
Authors: Zhan Bin , Ajmera Ravi , Geiger Stefan Michael , Gonçalves Marco Túlio Porto , Liu Zhuyun , Wei Junfei , Wilkins Patricia P. , Fujiwara Ricardo , Gazzinelli‐Guimaraes Pedro Henrique , Bottazzi Maria Elena , Hotez Peter
Published: October 2015

Objectives

To identify immunodominant antigens of Toxocara canis recognised by Toxocara‐infected sera as recombinant reagents for immunodiagnosis of toxocariasis.

Methods

Pooled sera from human cases of toxocariasis were used to identify immunodominant antigens by immunoscreening a T. canis larval expression cDNA library. The positive clones were sequenced to reveal the identity of the antigens. The recombinant proteins were expressed in E. coli and then used to confirm their immunoreaction with sera of humans with toxocariasis. Two chosen antigens were also used to differentiate Toxocara infection from other helminth infections in mice.

Results

Eleven antigens with immunodiagnostic potential were identified, including two C‐type lectins (CTLs) that reacted strongly with the Toxocara‐positive serum pool. The first CTL (Tc‐CTL‐1) is the same as TES‐32, previously identified as a major immunodominant component of TES; the second CTL (Tc‐CTL‐2) is a novel C‐type lectin sharing 83% amino acid sequence identity within the functional domain of Tc‐CTL‐1. The E. coli‐expressed recombinant Tc‐CTL‐1 was strongly recognised by the Toxocara‐positive serum pool or sera from animals experimentally infected with T. canis. Reactivity with recombinant Tc‐CTL‐1 was higher when the unreduced protein was used in an enzyme‐linked immunosorbent assay (ELISA), dot‐blot assay or Western blot test compared to the protein under reduced condition. Both recombinant Tc‐CTL‐1‐ and Tc‐CTL‐2‐based ELISAs were able to differentiate T. canis infection from other helminth infections in experimentally infected mice.

Conclusions

Both Tc‐CTL‐1 and Tc‐CTL‐2 were able to differentiate Toxocara infection from other helminth infections and could potentially be used as sensitive and specific immunodiagnostic antigens.
Outcome and Prognostic Factors for Canine Splenic Lymphoma Treated by Splenectomy (1995–2011)
Authors: van Stee Lucinda L. , Boston Sarah E. , Singh Ameet , Romanelli Giorgio , Rubio‐Guzman Alejandro , Scase Tim J.
Published: October 2015

Objective

To assess the outcome of canine splenic lymphoma treated with splenectomy and to evaluate prognostic factors, including involvement of other sites, adjuvant chemotherapy, and the effect of World Health Organization (WHO) histological classification of canine malignant lymphoma.

Design

Multi‐institutional, retrospective study.

Animals

Client‐owned dogs (n = 28).

Methods

Medical records (1995–2011) of dogs with a histological diagnosis of splenic lymphoma and treated by splenectomy submitted by Veterinary Society of Surgical Oncology members were reviewed. Included were dogs treated with or without adjuvant therapy. Overall survival, disease‐free interval, and cause of death were determined. Prognostic factors and the WHO histological classification of canine malignant lymphoma were evaluated with respect to outcome.

Results

Dogs with splenic lymphoma treated by splenectomy had a 1‐year survival rate of 58.8%, after which no animals died of their disease. B cell lymphoma held a better prognosis for survival than other variants of splenic lymphoma. Marginal zone lymphoma and mantle cell lymphoma were the most common B cell lymphoma subtypes in our study. Hemoabdomen and clinical signs related to splenic lymphoma, including abdominal distention, lethargy, and anorexia, were poor prognostic indicators, whereas disease confined to the spleen was a positive prognostic indicator. Pre‐ or postoperative adjuvant chemotherapy did not provide a survival benefit.

Conclusion

Based on our sample population, splenectomy alone was an effective treatment for splenic lymphoma in cases with disease confined to the spleen. Chemotherapy may not improve survival in cases of lymphoma restricted to the spleen.
Rad and Rem are non‐canonical G‐proteins with respect to the regulatory role of guanine nucleotide binding in CaV1.2 channel regulation
Authors: Chang Donald D. , Colecraft Henry M.
Published: October 2015

Key points

RGK (Rad, Rem, Rem2, Gem/Kir) proteins are small monomeric G‐proteins implicated in various cellular functions and disease states. All RGK proteins potently inhibit voltage‐gated calcium (CaV) channels.

It is unclear whether RGK proteins are regulated by guanine nucleotide binding in a manner that conforms to a canonical G‐protein regulation paradigm.

We utilized strategic Rad and Rem mutants together with a range of functional readouts (CaV1.2 currents, Ca2+ transients, CaVβ binding) in heterologous cells and cardiomyocytes to determine whether the function of these RGK proteins is regulated in a canonical manner.

Our results demonstrate that Rad and Rem are non‐canonical G‐proteins with respect to the regulatory role of their guanine nucleotide binding domain in CaV1.2 channel regulation.

Our findings offer deepened insights into cellular mechanisms governing RGK regulation and function, and contribute towards our understanding of their pathophysiological roles.

Rad and Rem are Ras‐like G‐proteins linked to diverse cardiovascular functions and pathophysiology. Understanding how Rad and Rem are regulated is important for deepened insights into their pathophysiological roles. As in other Ras‐like G‐proteins, Rad and Rem contain a conserved guanine‐nucleotide binding domain (G‐domain). Canonically, G‐domains are key control modules, functioning as nucleotide‐regulated switches of G‐protein activity. Whether Rad and Rem G‐domains conform to this canonical paradigm is ambiguous. Here, we used multiple functional measurements in HEK293 cells and cardiomyocytes (CaV1.2 currents, Ca2+ transients, CaVβ binding) as biosensors to probe the role of the G‐domain in regulation of Rad and Rem function. We utilized RadS105N and RemT94N, which are the cognate mutants to RasS17N, a dominant‐negative variant of Ras that displays decreased nucleotide binding affinity. In HEK293 cells, over‐expression of either RadS105N or RemT94N strongly inhibited reconstituted CaV1.2 currents to the same extent as their wild‐type (wt) counterparts, contrasting with reports that RadS105N is functionally inert in HEK293 cells. Adenovirus‐mediated expression of either wt Rad or RadS105N in cardiomyocytes dramatically blocked L‐type calcium current (ICa,L) and inhibited Ca2+‐induced Ca2+ release, contradicting reports that RadS105N acts as a dominant negative in heart. By contrast, RemT94N was significantly less effective than wt Rem at inhibiting ICa,L and Ca2+ transients in cardiomyocytes. FRET analyses in cardiomyocytes revealed that both RadS105N and RemT94N had moderately reduced binding affinity for CaVβs relative to their wt counterparts. The results indicate Rad and Rem are non‐canonical G‐proteins with respect to the regulatory role of their G‐domain in CaV1.2 regulation.
Function of the nucleus accumbens in motor control during recovery after spinal cord injury
Authors: Sawada Masahiro , Kato Kenji , Kunieda Takeharu , Mikuni Nobuhiro , Miyamoto Susumu , Onoe Hirotaka , Isa Tadashi , Nishimura Yukio
Published: October 2015

Motivation helps reverse neuronal damage

Motivation plays a fundamental role in mediating recovery after neurological injuries. Sawada
et al.
evaluated the potential contribution of the nucleus accumbens, a brain motivation center, to movement control after spinal cord injury in monkeys. The activity of the nucleus accumbens during the early phase after injury was important for the recovery of motor performance.

Science
, this issue p.
98
The promise of neurotechnology
Authors: Schwartz Andrew
Published: October 2015

The various brain initiatives for producing new tools, with the goal of advancing neurological therapies, have led to widespread attention on neurotechnology. Perhaps the most exciting aspect of this rapidly expanding field is the prospect of propelling experiments beyond scientific dogma to discover how the brain generates behavior. A sharper focus on this challenge will help direct the development of tools that can elucidate fundamental operations of the brain.
Cytomegalovirus IgG Level and Avidity in Breastfeeding Infants of HIV-Infected Mothers in Malawi
Authors: Kourtis Athena P. , Wiener Jeffrey , Chang Tiffany S. , Dollard Sheila C. , Amin Minal M. , Ellington Sascha , Kayira Dumbani , van der Horst Charles , Jamieson Denise J.
Published: October 2015

Cytomegalovirus (CMV) infection is common among infants of HIV-infected mothers in resource-limited settings. We examined the prevalence and timing of infant CMV infection during the first year of life using IgG antibody and avidity among HIV-exposed infants in Malawi and correlated the results with the presence of detectable CMV DNA in the blood. The Breastfeeding, Antiretrovirals and Nutrition (BAN) study randomized 2,369 mothers and their infants to maternal antiretrovirals, infant nevirapine, or neither for 28 weeks of breastfeeding, followed by weaning. Stored plasma specimens were tested for CMV IgG and antibody avidity from a random subset of infants who had been previously tested with blood CMV PCR and had available specimens at birth and at 24 and 48 weeks of age. Ninety-four of 127 infants (74.0%) tested at 24 weeks of age had CMV IgG of low or intermediate avidity, signifying primary CMV infections. An additional 22 infants (17.3%) had IgG of high avidity; 19 of them had CMV DNA detected in their blood, indicating infant infections. Taken together, these results show that the estimated prevalence of CMV infection at 24 weeks was 88.9%. By 48 weeks of age, 81.3% of infants had anti-CMV IgG; most of them (70.9%) had IgG of high avidity. The CMV serology and avidity testing, combined with the PCR results, confirmed a high rate of primary CMV infection by 6 months of life among breastfeeding infants of HIV-infected mothers. The CMV PCR in blood detected most, but not all, infant CMV infections.
Simplified Protocol for Carba NP Test for Enhanced Detection of Carbapenemase Producers Directly from Bacterial Cultures
Authors: Pasteran Fernando , Tijet Nathalie , Melano Roberto G. , Corso Alejandra
Published: October 2015

We compared carbapenemase detection among 266 Gram-negative bacilli (161 carbapenemase producers) using the Carba NP tests issued by the CLSI (CNPt-CLSI) and a novel protocol (CNPt-direct) designed for carbapenemase detection direct from bacterial cultures (instead of bacterial extracts required by the CLSI tests). The specificities were comparable (100%), but the CNPt-direct was more sensitive (98% versus 84%). The CNPt-direct was easier to perform due to the direct use of colonies and offered a more robust detection of carbapenemase producers.
Flavivirus Infection Impairs Peroxisome Biogenesis and Early Antiviral Signaling
Authors: You Jaehwan , Hou Shangmei , Malik-Soni Natasha , Xu Zaikun , Kumar Anil , Rachubinski Richard A. , Frappier Lori , Hobman Tom C.
Published: October 2015

Flaviviruses are significant human pathogens that have an enormous impact on the global health burden. Currently, there are very few vaccines against or therapeutic treatments for flaviviruses, and our understanding of how these viruses cause disease is limited. Evidence suggests that the capsid proteins of flaviviruses play critical nonstructural roles during infection, and therefore, elucidating how these viral proteins affect cellular signaling pathways could lead to novel targets for antiviral therapy. We used affinity purification to identify host cell proteins that interact with the capsid proteins of West Nile and dengue viruses. One of the cellular proteins that formed a stable complex with flavivirus capsid proteins is the peroxisome biogenesis factor Pex19. Intriguingly, flavivirus infection resulted in a significant loss of peroxisomes, an effect that may be due in part to capsid expression. We posited that capsid protein-mediated sequestration and/or degradation of Pex19 results in loss of peroxisomes, a situation that could result in reduced early antiviral signaling. In support of this hypothesis, we observed that induction of the lambda interferon mRNA in response to a viral RNA mimic was reduced by more than 80%. Together, our findings indicate that inhibition of peroxisome biogenesis may be a novel mechanism by which flaviviruses evade the innate immune system during early stages of infection.

IMPORTANCE
RNA viruses infect hundreds of millions of people each year, causing significant morbidity and mortality. Chief among these pathogens are the flaviviruses, which include dengue virus and West Nile virus. Despite their medical importance, there are very few prophylactic or therapeutic treatments for these viruses. Moreover, the manner in which they subvert the innate immune response in order to establish infection in mammalian cells is not well understood. Recently, peroxisomes were reported to function in early antiviral signaling, but very little is known regarding if or how pathogenic viruses affect these organelles. We report for the first time that flavivirus infection results in significant loss of peroxisomes in mammalian cells, which may indicate that targeting of peroxisomes is a key strategy used by viruses to subvert early antiviral defenses.
Comparison of the Life Cycles of Genetically Distant Species C and Species D Human Adenoviruses Ad6 and Ad26 in Human Cells
Authors: Turner Mallory A. , Middha Sumit , Hofherr Sean E. , Barry Michael A.
Published: October 2015

Our understanding of adenovirus (Ad) biology is largely extrapolated from human species C Ad5. Most humans are immune to Ad5, so lower-seroprevalence viruses like human Ad6 and Ad26 are being tested as therapeutic vectors. Ad6 and Ad26 differ at the DNA level by 34%. To better understand how this might impact their biology, we examined the life cycle of the two viruses in human lung cells
in vitro
. Both viruses infected A549 cells with similar efficiencies, executed DNA replication with identical kinetics within 12 h, and began killing cells within 72 h. While Ad6-infected cells remained adherent until death, Ad26-infected cells detached within 12 h of infection but remained viable. Next-generation sequencing (NGS) of mRNA from infected cells demonstrated that viral transcripts constituted 1% of cellular mRNAs within 6 h and 8 to 16% within 12 h. Quantitative PCR and NGS revealed the activation of key early genes at 6 h and transition to late gene activation by 12 h by both viruses. There were marked differences in the balance of E1A and E1B activation by the two viruses and in the expression of E3 immune evasion mRNAs. Ad6 was markedly more effective at suppressing major histocompatibility complex class I (MHC I) display on the cell surface and in evading TRAIL-mediated apoptosis than was Ad26. These data demonstrate shared as well as divergent life cycles in these genetically distant human adenoviruses. An understanding of these differences expands the knowledge of alternative Ad species and may inform the selection of related Ads for therapeutic development.

IMPORTANCE
A burgeoning number of adenoviruses (Ads) are being harnessed as therapeutics, yet the biology of these viruses is generally extrapolated from Ad2 and Ad5. Here, we are the first to compare the transcriptional programs of two genetically distant Ads by mRNA next-generation sequencing (NGS). Species C Ad6 and Ad26 are being pursued as lower-seroprevalence Ad vectors but differ at the DNA level by 34%. Head-to-head comparison in human lung cells by NGS revealed that the two viruses generally conform to our general understanding of the Ad transcriptional program. However, fine mapping revealed subtle and strong differences in how these two viruses execute these programs, including differences in the balance of E1A and E1B mRNAs and in E3 immune evasion genes. This suggests that not all adenoviruses behave like Ad2 and Ad5 and that they may have unique strategies to infect cells and evade the immune system.
ras oncogene activation of a VL30 transcriptional element is linked to transformation.
Authors: Owen R D , Bortner D M , Ostrowski M C
Published: October 2015

The activity of a murine VL30 transcriptional element was increased 20-fold in transient assays by coexpression of mutant ras genes. The cis element did not respond to ras in a revertant cell line that was transformation defective. Therefore, ras-dependent alterations in transcription and ras transformation are linked. Deletion analysis of the VL30 long-terminal-repeat U3 region showed that a minimal 53-base-pair segment is required in cis for oncogene activation of transcription. Gel retention assays using a probe that contained the minimal cis element revealed that a unique complex was formed with nuclear proteins prepared from transformed cells. Exonuclease III footprinting and gel retention experiments that used oligonucleotide probes and competitors indicated that two distinct nuclear factors interact with the minimal cis-responsive element. Site-directed deletion of the 5'-proximal binding site (TGACTCT) resulted in a complete loss of ras responsiveness. However, deletion of this site did not affect stimulation by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). These data are consistent with the hypothesis that ras and TPA signal transduction mechanisms for transcriptional activation are distinct.
Intermolecular recombination between DNAs introduced into mouse L cells is mediated by a nonconservative pathway that leads to crossover products.
Authors: Lin F L , Sperle K , Sternberg N
Published: October 2015

We describe experiments designed to measure the efficiency of intermolecular recombination between mutant herpesvirus thymidine kinase (tk) genes introduced into mouse L cells. Recombinants were scored as stable transformants containing a functional tk gene. The two recombination substrates used were ptkB8, a pBR322-based plasmid containing a mutant tk gene, with a BamHI linker in an SphI restriction site that is centrally located within the gene, and mp10tk delta 3' delta 5', an mp10 vector with a tk gene deleted at both the 3' and 5' ends. The only homology shared by the two DNAs is 885 base pairs within the tk gene. To determine whether the double-strand break repair model that has been used to explain recombination in yeast cells (J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, and F. W. Stahl, Cell 33:25-35, 1983) can account for recombination during the introduction of these DNAs into mammalian cells, we transformed cells with BamHI-linearized ptkB8 and supercoiled mp10tk delta 3' delta 5' replicative-form DNA. These two DNAs should recombine efficiently according to that model and should generate gene conversion products. In this reaction, the supercoiled DNA acts as the donor of information to repair the cleaved tk gene. Our results indicated that the efficiency of this reaction was very low (less than 10 transformants were obtained per 0.1 microgram of each DNA used in the reaction per 10(6) cells). In contrast, if BamHI-cleaved ptkB8 DNA was cotransformed into cells along with a circular DNA molecule containing a tk gene deleted only at its 3' end or only at its 5' end (mp10tk delta 3' or mp10tk delta 5'), then the efficiency of recombination could be more than 4 orders of magnitude higher than it was with circular mp10tk delta 3' delta 5' DNA. Recombination frequencies were highest when the tk delta 3' or tk delta 5' DNA used was cleaved at the tk deletion junction. Southern analyses of DNA from TK+ transformants generated with BamHI-cleaved ptkB8 and BamHI-cleaved mp10tk delta 3' DNAs indicated that recombination was almost always associated with the reassortment of markers flanking the reconstructed tk DNA. Together, these results are more consistent with the nonconservative single-strand annealing model for recombination that we proposed several years ago (F.-L. Lin, K. Sperle, and N. Sternberg, Mol. Cell. Biol. 4:1020-1034, 1984) than they are with the double-strand break repair model.
PRP18, a protein required for the second reaction in pre-mRNA splicing.
Authors: Vijayraghavan U , Abelson J
Published: October 2015

We have investigated the role of a novel temperature-sensitive splicing mutation, prp18. We had previously demonstrated that an accumulation of the lariat intermediate of splicing occurred at the restrictive temperature in vivo. We have now used the yeast in vitro splicing system to show that extracts from this mutant strain are heat labile for the second reaction of splicing. The heat inactivation of prp18 extracts results from loss of activity of an exchangeable component. Inactivated prp18 extracts are complemented by heat-inactivated extracts from other mutants or by fractions from wild-type extracts. In heat-inactivated prp18 extracts, 40S splicing complexes containing lariat intermediate and exon 1 can assemble. The intermediates in this 40S complex can be chased to products by complementing extracts in the presence of ATP. Both complementation of extracts and chasing of the isolated prp18 spliceosomes takes place with micrococcal nuclease-treated extracts. Furthermore, the complementation profile with fractions of wild-type extracts indicates that the splicing defect results from a mutation in a previously designated factor required for the second step of splicing. The isolation of this mutant as temperature-sensitive lethal has also facilitated cloning of the wild-type allele by complementation.
Diacylglycerol production in Xenopus laevis oocytes after microinjection of p21ras proteins is a consequence of activation of phosphatidylcholine metabolism.
Authors: Lacal J C
Published: October 2015

Microinjection of p21Ha-ras proteins into Xenopus laevis oocytes induces a rapid increase of 1,2-diacylglycerol (DAG) levels. The observed alterations in DAG levels were consistent with the ability of the protein to induce maturation, measured by germinal vesicle breakdown (GVBD). Both the increase in DAG levels and GVBD activity were dependent on the ability of the proteins to undergo membrane translocation. Alterations of DAG levels or GVBD activity did not correlate with changes in the levels of inositol phosphates. However, at minimal doses sufficient to achieve maximal biological response, a biphasic increase in the amounts of phosphocholine and CDP-choline was observed. The first burst of phosphocholine and CDP-choline preceded the increase in DAG levels. The second peak paralleled the appearance of DAG. Choline kinase activity was also increased in oocyte extracts after p21ras microinjection. These results suggest that both the synthesis and degradation of phosphatidylcholine are activated after microinjection of ras proteins into Xenopus oocytes, resulting in a net production of DAG.
Functional analysis of the murine T-cell receptor beta enhancer and characteristics of its DNA-binding proteins.
Authors: Takeda J , Cheng A , Mauxion F , Nelson C A , Newberry R D , Sha W C , Sen R , Loh D Y
Published: October 2015

The minimal T-cell receptor (TCR) beta-chain (TCR beta) enhancer has been identified by transfection into lymphoid cells. The minimal enhancer was active in T cells and in some B-lineage cells. When a larger fragment containing the minimal enhancer was used, its activity was apparent only in T cells. Studies with phytohemagglutinin and 4 beta-phorbol-12,13-dibutyrate revealed that the enhancer activity was increased by these agents. By a combination of DNase I footprinting, gel mobility shift assay, and methylation interference analysis, seven different motifs were identified within the minimal enhancer. Furthermore, competition experiments showed that some of these elements bound identical or similar factors that are known to bind to the TCR V beta promoter decamer or to the immunoglobulin enhancer kappa E2 or muEBP-E motif. These shared motifs may be important in the differential gene activity among the different lymphoid subsets.
Gene induction by interferons: functional complementation between trans-acting factors induced by alpha interferon and gamma interferon.
Authors: Bandyopadhyay S K , Kalvakolanu D V , Sen G C
Published: October 2015

HeLaM is a variant cell line in which the transcriptional induction of many genes by alpha interferon has special characteristics (Tiwari et al., Mol. Cell. Biol. 8:4289-4294, 1988). The same characteristics were also displayed for induced transcription of a permanently transfected chimeric gene containing the interferon-stimulated response element of gene 561. For understanding the molecular basis of the special requirements of HeLaM cells, an analysis of the interferon-stimulated gene factors (ISGF) was undertaken. By using gel shift assays, it was shown that the activation of ISGF3 by alpha interferon treatment of HeLaM cells had characteristics identical to those of induced transcription: inhibition by 2-aminopurine and the need for ongoing protein synthesis which was obviated by pretreating the cells with gamma interferon. Upon mixing in vitro the cytoplasmic fraction of gamma interferon-treated HeLaM cells with that of cells treated with alpha interferon and cycloheximide, active ISGF3 was reconstituted, presumably through complementation of two components, ISGF3 gamma and ISGF3 alpha, present in the two respective fractions. Because, unlike other cells, untreated HeLaM cells did not contain detectable levels of either component, we could induce them individually and study their independent properties. Induction of ISGF3 gamma but not of ISGF3 alpha needed ongoing protein synthesis and was blocked by 2-aminopurine. Once induced, ISGF3 gamma was active for 24 h and was present in both the nuclear and cytoplasmic fractions. Activated ISGF3 alpha, on the other hand, did not translocate to the nucleus in the absence of ISGF3 gamma, and in the cytoplasm its activity decayed within 2 h of its activation. In reference to our working model, all of the above observations indicate that ISGF3 gamma is the product of signal 1 and ISGF3 alpha is the product of signal 2.
E4F and ATF, two transcription factors that recognize the same site, can be distinguished both physically and functionally: a role for E4F in E1A trans activation.
Authors: Rooney R J , Raychaudhuri P , Nevins J R
Published: October 2015

Previous experiments have identified an element in the adenovirus E4 promoter that is critical for E1A-dependent trans activation and that can confer inducibility to a heterologous promoter. This DNA element is a recognition site for multiple nuclear factors, including ATF, which is likely a family of DNA-binding factors with similar DNA recognition properties. However, ATF activity was found not to be altered in any demonstrable way as a result of adenovirus infection. In contrast, another factor that recognizes this element, termed E4F, was found at only very low levels in uninfected cells but was increased markedly upon adenovirus infection, as measured in DNA-binding assays. Although both the ATF activity and the E4F activity recognized and bound to the same two sites in the E4 promoter, they differed in their sequence recognition of these sites. Furthermore, E4F bound only to a small subset of the ATF recognition sites; for instance, E4F did not recognize the ATF sites in the E2 or E3 promoters. Various E4F and ATF binding sites were inserted into an expression vector and tested by cotransfection assays for responsiveness to E1A. We found that a sequence capable of binding E4F could confer E1A inducibility. In contrast, a sequence that could bind ATF but not E4F did not confer E1A inducibility. We also found that E4F formed a stable complex with the E4 promoter, whereas the ATF DNA complex was unstable and rapidly dissociated. We conclude that the DNA-binding specificity of E4F as well as the alterations in DNA-binding activity of E4F closely correlates with E1A stimulation of the E4 promoter.
Characterization of promoter elements of an interferon-inducible Ly-6E/A differentiation antigen, which is expressed on activated T cells and hematopoietic stem cells.
Authors: Khan K D , Lindwall G , Maher S E , Bothwell A L
Published: October 2015

The Ly-6E/A antigen is expressed on activated murine T cells. Using probes made from the previously characterized cDNA, we have isolated a genomic DNA clone encoding the Ly-6A antigen. We determined the DNA sequence of the genomic clone and conducted a functional analysis of the promoter region. Mouse fibroblast BALB/3T3 cells transfected with this genomic clone constitutively expressed Ly-6A antigen on their cell surface. This expression was inducible by alpha/beta and gamma interferons. The Ly-6E 5'-flanking region was analyzed by chloramphenicol acetyltransferase assays in fibroblast cells for cis-acting elements. At least two positive elements were found to be needed for maximum constitutive promoter activity in L cells. One of the positive elements was specifically bound by a CCAAT box-binding protein from crude nuclear extract, as shown by electrophoretic mobility shift assays and footprinting. The other element, which contains a GGAAA motif and has homology to various known enhancers, also showed a specific binding activity. This second positive element when multimerized became a very powerful enhancing element. Interferon treatment could enhance expression of the chloramphenicol acetyltransferase gene fused to the Ly-6E 5'-flanking region in stably transfected BALB/3T3 cells. The elements responsible for this enhancement lie, at least in part, between positions -1760 and -900 of the gene. Surprisingly, there is no sequence homology between this region of Ly-6E and the established consensus for the interferon-stimulated response element, which has been shown functionally important to all previously characterized alpha/beta interferon-inducible promoters. The Ly-6E gene may prove to be a novel system for the study of interferon induction.
Activation of p56lck through mutation of a regulatory carboxy-terminal tyrosine residue requires intact sites of autophosphorylation and myristylation.
Authors: Abraham N , Veillette A
Published: October 2015

Mutation of the major site of in vivo tyrosine phosphorylation of p56lck (tyrosine 505) to a phenylalanine constitutively enhances the p56lck-associated tyrosine-specific protein kinase activity. The mutant polypeptide is extensively phosphorylated in vivo at the site of in vitro Lck autophosphorylation (tyrosine 394) and is capable of oncogenic transformation of rodent fibroblasts. These observations have suggested that phosphorylation at Tyr-505 down regulates the tyrosine protein kinase activity of p56lck. Herein we have attempted to examine whether other posttranslational modifications may be involved in regulation of the enzymatic function of p56lck. The results indicated that activation of p56lck by mutation of Tyr-505 was prevented by a tyrosine-to-phenylalanine substitution at position 394. Furthermore, activation of p56lck by mutation of the carboxy-terminal tyrosine residue was rendered less efficient by substituting an alanine residue for the amino-terminal glycine. This second mutation prevented p56lck myristylation and stable membrane association and was associated with decreased in vivo phosphorylation at Tyr-394. Taken together, these findings imply that lack of phosphorylation at Tyr-505 may be insufficient for enhancement of the p56lck-associated tyrosine protein kinase activity. Our data suggest that activation of p56lck may be dependent on phosphorylation at Tyr-394 and that this process may be facilitated by myristylation, membrane association, or both.
Molecular cloning and analysis of the scon-2 negative regulatory gene of Neurospora crassa.
Authors: Paietta J V
Published: October 2015

The sulfur regulatory system of Neurospora crassa is composed of a group of highly regulated structural genes (e.g., the gene encoding arylsulfatase) that are under coordinate control of scon+ (sulfur controller) negative and cys-3+ positive regulatory genes. In scon-1 (previously designated sconC) and scon-2 mutants, there is constitutive expression of sulfur structural genes regardless of the sulfur level available to the cells. The scon-2+ gene was cloned by sib selection screening of a cosmid-based gene library. The screening was based on the use of chromate, a toxic sulfate analog, which is transported into scon-2 cells grown on high sulfur but is not transported into cells that have regained normal sulfur regulation. Restriction fragment length polymorphism analysis was used to confirm that the cloned segment mapped to the proper chromosomal location. In wild-type cells, Northern (RNA) blot analysis showed that a 2.6-kilobase scon-2+ transcript was present at a substantial level only under sulfur-derepressing conditions. Kinetic analysis showed that scon-2+ mRNA content increased as the cells became sulfur starved. Further, scon-2+ RNA was detectable in a nuclear transcription assay only under derepressing conditions. In scon-1, the levels of scon-2+ mRNA were found to be constitutive. In the cys-3 regulatory mutant, there was a reduced level of scon-2+ transcript. cys-3+ and ars-1+ mRNAs were present under both derepressing and repressing conditions in the scon-2 mutant. Repeat-induced point mutation-generated scon-2 mutants were identical in phenotype to the known mutant.
Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae.
Authors: Baronas-Lowell D M , Warner J R
Published: October 2015

In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.
Sequence conservation and structural organization of the glycerol-3-phosphate dehydrogenase promoter in mice and humans.
Authors: Gwynn B , Lyford K A , Birkenmeier E H
Published: October 2015

Cloned segments of the mouse glycerol-3-phosphate dehydrogenase (GPDH) gene, Gdc-1, were used to screen a human library. Human clones obtained spanned 25 kilobases of genomic DNA containing the human GPDH gene, GPD1. The 4 kb of sequence obtained from the 5'-flanking region and first exon of GPD1 was compared with the corresponding mouse sequence. Both sequences share a HindIII site located in what has proven to be the highly conserved 3' untranslated region of an upstream gene of unknown function, D15Kzl. The 3.6-kilobase segment of mouse DNA located between D15Kzl and Gdc-1 was provisionally termed the GPDH promoter. Alignment of the mouse promoter with the corresponding human sequence revealed two conserved domains. An upstream distal promoter region is approximately 900 base pairs in length. A downstream or proximal promoter region consists of approximately 300 base pairs immediately upstream of a TATA-like box and contains the fat-specific elements 1 and 2. Analysis of the chromatin structure of the Gdc-1 promoter revealed four DNase I-hypersensitive sites. They were present in DNA of liver and brown fat, in which GPDH expression is high, but were absent in DNA of spleen, in which GPDH expression is low. Methylation studies of the promoter showed it to be heavily methylated in sperm. However, the DNA from each adult somatic tissue had a unique distribution of nonmethylated sites and could easily be identified by its methylation pattern. These data suggest a structural model of the promoter that explains how Gdc-1 expression is differentially regulated in many types of cells.