bluuebunny/crossref_metadata_2025 huggingface dataset:
Sodium nitrate alleviates functional muscle ischaemia in patients with Becker muscular dystrophy
Authors: Nelson Michael D. , Rosenberry Ryan , Barresi Rita , Tsimerinov Evgeny I. , Rader Florian , Tang Xiu , Mason O'Neil , Schwartz Avery , Stabler Thomas , Shidban Sarah , Mobaligh Neigena , Hogan Shomari , Elashoff Robert , Allen Jason D. , Victor Ronald G.
Published: October 2015

Key points

Dystrophin deficiency disrupts sarcolemmal targeting of neuronal nitric oxide synathase, resulting in functional muscle ischaemia.

Chronic treatment of dystrophic mice with an inorganic nitric oxide (NO) donor alleviates this ischaemia and improves many features of the dystrophic phenotype.

The present study translates this preclinical work by showing that a single oral dose of sodium nitrate,which serves as a NO donor when reduced to circulating nitrite by the commensal bacteria in the oral cavity, alleviates functional muscle ischaemia and restores normal blood flow regulation in human patients with dystrophinopathy.

The results of the present study further support the mechanistic hypothesis that circulating nitrite serves as an alternative NO donor when reduced by deoxyhaemoglobin and/or deoxymyoglobin in exercising muscle.

Becker muscular dystrophy (BMD) is a progressive X‐linked muscle wasting disease for which there is no treatment. BMD is caused by in‐frame mutations in the gene encoding dystrophin, a structural cytoskeletal protein that also targets other proteins to the sarcolemma. Among these is neuronal nitric oxide synthase mu (nNOSμ), which requires specific spectrin‐like repeats (SR16/17) in dystrophin's rod domain and the adaptor protein α‐syntrophin for sarcolemmal targeting. When healthy skeletal muscle is exercised, sarcolemmal nNOSμ‐derived nitric oxide (NO) attenuates α‐adrenergic vasoconstriction, thus optimizing perfusion. In the mdx mouse model of dystrophinopathy, this protective mechanism (functional sympatholysis) is defective, resulting in functional muscle ischaemia. Treatment with a NO‐donating non‐steroidal anti‐inflammatory drug (NSAID) alleviates this ischaemia and improves the murine dystrophic phenotype. In the present study, we report that, in 13 men with BMD, sympatholysis is defective mainly in patients whose mutations disrupt sarcolemmal targeting of nNOSμ, with the vasoconstrictor response measured as a decrease in muscle oxygenation (near infrared spectroscopy) to reflex sympathetic activation. Then, in a single‐arm, open‐label trial in 11 BMD patients and a double‐blind, placebo‐controlled cross‐over trial in six patients, we show that acute treatment with oral sodium nitrate, an inorganic NO donor without a NSIAD moiety, restores sympatholysis and improves post‐exercise hyperaemia (Doppler ultrasound). By contrast, sodium nitrate improves neither sympatholysis, nor hyperaemia in healthy controls. Thus, a simple NO donor recapitulates the vasoregulatory actions of sarcolemmal nNOS in BMD patients, and constitutes a putative novel therapy for this disease.
Incorporation of nanostructured hydroxyapatite and poly(N-isopropylacrylamide) in demineralized bone matrix enhances osteoblast and human mesenchymal stem cell activity
Authors: Nicoletti Alessandro , Torricelli Paola , Bigi Adriana , Fornasari Piermaria , Fini Milena , Moroni Lorenzo
Published: October 2015

Demineralized bone matrix (DBM) is currently used in many clinical applications for bone augmentation and repair. DBM is normally characterized by the presence of bone morphogenetic proteins. In this study, the authors have optimized methods to obtain DBM under good manufacturing practice, resulting in enhanced bioactivity. The processed DBM can be used alone, together with nanostructured hydroxyapatite (nanoHA), or dispersed in a physiological carrier or hydrogel. In this study, osteoblasts (MG-63) and human bone marrow derived mesenchymal stem cells (hMSCs) were cultured on DBM pastes made in phosphate buffered saline solution or poly(N-isopropylacrylamide) (PNIPAAM) hydrogels with or without nanoHA. The authors observed that the presence of PNIPAAM reduced osteoblast adhesion, while the addition of nanoHA increased osteoblast adhesion, proliferation, interleukin-6 (IL-6) production, and reduced lactate dehydrogenase (LDH) production. Increasing concentrations of PNIPAAM in combination with nanoHA further increased osteoblast proliferation, and decreased IL-6 and LDH production. Incorporation of PNIPAAM in DBM enhanced hMSCs proliferation and collagen type-I production. Furthermore, a combination of PNIPAAM and nanoHA further increased alkaline phosphatase and osteocalcin production in hMSCs, independently from the concentration of PNIPAAM. This study shows that combinations of DBM with nanoHA and PNIPAAM seem to offer a promising route to enhance cell activity and induce osteogenic differentiation.
Modification of porous SiOCH by first contact with water vapor after plasma process
Authors: Darnon Maxime , Rochat Névine , Licitra Christophe
Published: October 2015

Porous low-k dielectrics used in integrated circuits interconnects are sensitive to plasma processes and exposure to moist ambient. In this paper, the authors use a vacuum and dry N2 sample transportation in conjunction with controlled atmosphere infrared spectroscopy (in transmission and in multiple internal reflections mode) to investigate the role of the first exposure to humidity on plasma-damaged porous low-k. They show that a porous low-k exposed to a N2/H2 or O2 plasma is irreversibly modified by the first exposure to water molecules. In the former case, Si-NHx and Si-H groups are converted into Si-OH while C=O and/or CH=NH groups are formed. In the latter case, irreversible water uptake as icelike water leads to a decrease of the density of isolated silanol groups. In both cases, no additional modifications are observed after a second cycle of water adsorption/desorption, meaning that subsequent water adsorption is reversible.
Highly selective removal of poly(methyl methacrylate) from polystyrene-block-poly(methyl methacrylate) by CO/H2 plasma etching
Authors: Imamura Tsubasa , Yamamoto Hiroshi , Omura Mitsuhiro , Sakai Itsuko , Hayashi Hisataka
Published: October 2015

The directed self-assembly lithography process using polystyrene (PS)-block-poly(methyl methacrylate) (PMMA) requires selective removal of PMMA, which is called the development process. The development process using plasma etching (dry development) without surface roughness of the line/space pattern was investigated. First, the authors focused on the chemical compositions of PMMA and PS. Using CO plasma, highly selective etching of PMMA was achieved (PMMA/PS etch selectivity >20). It was found that the PS surface roughness induced by plasma treatment depended on the thickness of the deposition layer formed on the PS surface. To suppress the PS surface roughness because of plasma treatment, the authors controlled the thickness of the deposition layer by adding H2 gas to the CO plasma. Using CO/H2 plasma, highly selective PMMA/PS etching without surface roughness was achieved. Consequently, the authors successfully achieved dry development of random lamella patterns by application of the CO/H2 plasma process.
Maximal, Not Habitual, Walking Velocity is More Highly Correlated to Functional Fitness of Community-Dwelling Older Adults
Authors: Gray Michelle , Paulson Sally , Powers Melissa
Published: October 2015

The aim of this investigation was to determine the relationship between usual and maximal walking velocities with measures of functional fitness (FF). Fifty-seven older adults (78.2 ± 6.6 years) were recruited from a local retirement community. All participants completed the following assessments: 10-m usual and maximal walk, Short Physical Performance Battery (SPPB), 6-min walk (6MW), 8-foot up-and-go (UPGO), and 30-s chair stand. Based on their SPPB performance, low (≤ 9) and high (≥ 10) FF groups were formed. Among all participants, maximal walking velocity, not usual walking velocity, was significantly correlated with SPPB (r = .35; p < .05 and r = .19; p > .05, respectively). In the high functioning group, both maximal and usual walking velocities were correlated, but correlation coefficients were stronger for all variables for maximal walking velocity. These results suggest different walking conditions may be necessary to use for high and low functioning older adults; specifically, maximal walking velocity may be a preferred measure among high functioning older adults.
Programmed death of bacterial spores
Authors: VanHook Annalisa M.
Published: October 2015

CmpA functions in a quality-control checkpoint that ensures that only properly formed bacterial spores are released into the environment.
Inhibiting one kinase to kill them all
Authors: Ferrarelli Leslie K.
Published: October 2015

Inhibiting a single kinase that promotes transcription may kill highly heterogenic breast tumors.
Esters of Pyrazinoic Acid Are Active against Pyrazinamide-Resistant Strains of Mycobacterium tuberculosis and Other Naturally Resistant Mycobacteria
In Vitro
and
Ex Vivo
within Macrophages
Authors: Pires David , Valente Emília , Simões Marta Filipa , Carmo Nuno , Testa Bernard , Constantino Luís , Anes Elsa
Published: October 2015

Pyrazinamide (PZA) is active against major
Mycobacterium tuberculosis
species (
M. tuberculosis
,
M. africanum
, and
M. microti
) but not against
M. bovis
and
M. avium
. The latter two are mycobacterial species involved in human and cattle tuberculosis and in HIV coinfections, respectively. PZA is a first-line agent for the treatment of human tuberculosis and requires activation by a mycobacterial pyrazinamidase to form the active metabolite pyrazinoic acid (POA). As a result of this mechanism, resistance to PZA, as is often found in tuberculosis patients, is caused by point mutations in pyrazinamidase. In previous work, we have shown that POA esters and amides synthesized in our laboratory were stable in plasma (M. F. Simões, E. Valente, M. J. Gómez, E. Anes, and L. Constantino, Eur J Pharm Sci 37:257–263, 2009,
http://dx.doi.org/10.1016/j.ejps.2009.02.012
). Although the amides did not present significant activity, the esters were active against sensitive mycobacteria at concentrations 5- to 10-fold lower than those of PZA. Here, we report that these POA derivatives possess antibacterial efficacy
in vitro
and
ex vivo
against several species and strains of
Mycobacterium
with natural or acquired resistance to PZA, including
M. bovis
and
M. avium
. Our results indicate that the resistance probably was overcome by cleavage of the prodrugs into POA and a long-chain alcohol. Although it is not possible to rule out that the esters have intrinsic activity
per se
, we bring evidence here that long-chain fatty alcohols possess a significant antimycobacterial effect against PZA-resistant species and strains and are not mere inactive promoieties. These findings may lead to candidate dual drugs having enhanced activity against both PZA-susceptible and PZA-resistant isolates and being suitable for clinical development.
Clinical Pharmacokinetics of Rifampin in Patients with Tuberculosis and Type 2 Diabetes Mellitus: Association with Biochemical and Immunological Parameters
Authors: Medellín-Garibay S. E. , Cortez-Espinosa N. , Milán-Segovia R. C. , Magaña-Aquino M. , Vargas-Morales J. M. , González-Amaro R. , Portales-Pérez D. P. , Romano-Moreno S.
Published: October 2015

Tuberculosis (TB) remains a major public health issue due to the increasing incidence of type 2 diabetes mellitus (T2DM), which exacerbates the clinical course of TB and increases the risk of poor long-term outcomes. The aim of this study was to characterize the pharmacokinetics of rifampin (RIF) and its relationship with biochemical and immunological parameters in patients with TB and T2DM. The biochemical and immunological parameters were assessed on the same day that the pharmacokinetic evaluation of RIF was performed. Factors related to the metabolic syndrome that is characteristic of T2DM patients were not detected in the TB-T2DM group (where predominant malnutrition was present) or in the TB group. Percentages of CD8
+
T lymphocytes and NK cells were diminished in the TB and TB-T2DM patients, who had high tumor necrosis factor alpha (TNF-α) and low interleukin-17 (IL-17) levels compared to healthy volunteers. Delayed RIF absorption was observed in the TB and TB-T2DM patients; absorption was poor and slower in the latter group due to poor glycemic control. RIF clearance was also slower in the diabetic patients, thereby prolonging the mean residence time of RIF. There was a significant association between glycemic control, increased TNF-α serum concentrations, and RIF pharmacokinetics in the TB-T2DM patients. These altered metabolic and immune conditions may be factors to be considered in anti-TB therapy management when TB and T2DM are concurrently present.
Susceptibility Pattern of Microorganisms Isolated by Percutaneous Needle Biopsy in Nonbacteremic Pyogenic Vertebral Osteomyelitis
Authors: Desoutter Sophie , Cottier Jean-Philippe , Ghout Idir , Issartel Bertrand , Dinh Aurélien , Martin Arnaud , Carlier Robert , Bernard Louis ,   the Duration of Treatment for Spondylodiscitis Study Group
Published: October 2015

Pyogenic vertebral osteomyelitis (VO) is diagnosed according to several lines of evidence: clinical, biological, radiological, and histological. Definitive diagnosis requires the isolation of a causative pathogen or histological confirmation. The aim of our study was to describe the microorganisms isolated by percutaneous needle biopsy (PNB) and to analyze their susceptibility patterns, in order to assess the possibility of empirical combination therapy for the treatment of nonbacteremic patients without resorting to PNB. Based on a French prospective multicenter study of 351 patients with VO, we compiled clinical, biological, and radiological findings for 101 patients with microbiologically confirmed VO. Based on antibiotic susceptibility testing of PNB isolated pathogens, the suitabilities of four antibiotic combinations were analyzed: ofloxacin plus rifampin, levofloxacin plus rifampin, ciprofloxacin plus clindamycin, and ciprofloxacin plus amoxicillin-clavulanate. The main causative pathogens identified were coagulase-negative
Staphylococcus
spp. (26% of isolates), followed by
Staphylococcus aureus
(21%),
Streptoccocus
spp. (13%), and enterobacteria (21%). Empirical antibiotic combination therapy was effective in nearly 75% of cases, and the different combinations gave similar results, except for ofloxacin-rifampin, which was effective in only 58% of cases. A “perfect” empirical antibiotic therapy does not exist. If PNB is not possible, a combination of a fluoroquinolone with clindamycin or rifampin can be used, but the high risk of microbiological failure does not allow the exclusion of PNB. (This study has been registered with EudraCT, number 2006-000951-18, and ClinicalTrials.gov, number NCT00764114.)
Increased Hydrolysis of Oximino-β-Lactams by CMY-107, a Tyr199Cys Mutant Form of CMY-2 Produced by Escherichia coli
Authors: Kotsakis S. D. , Miriagou V. , Vetouli E. E. , Bozavoutoglou E. , Lebessi E. , Tzelepi E. , Tzouvelekis L. S.
Published: October 2015

The cephalosporinase CMY-107, a Tyr199Cys mutant form of CMY-2 encoded by an IncI self-transferable plasmid carried by an
Escherichia coli
clinical strain, was characterized. The enzyme hydrolyzed oximino-cephalosporins and aztreonam more efficiently than CMY-2 did.
In Vitro
Activity of Isavuconazole and Comparators against Clinical Isolates of the
Mucorales
Order
Authors: Arendrup Maiken Cavling , Jensen Rasmus Hare , Meletiadis Joseph
Published: October 2015

The
in vitro
activity of isavuconazole against
Mucorales
isolates measured by EUCAST E.Def 9.2 and CLSI M38-A2 methodologies was investigated in comparison with those of amphotericin B, posaconazole, and voriconazole. Seventy-two isolates were included: 12 of
Lichtheimia corymbifera
, 5 of
Lichtheimia ramosa
, 5 of group I and 9 of group II of
Mucor circinelloides
, 9 of
Rhizomucor pusillus
, 26 of
Rhizopus microsporus
, and 6 of
Rhizopus oryzae
. Species identification was confirmed by internal transcribed spacer (ITS) sequencing. EUCAST MICs were read on day 1 (EUCAST-d1) and day 2 (EUCAST-d2), and CLSI MICs were read on day 2 (CLSI-d2). Isavuconazole MIC
50
s (range) (mg/liter) by EUCAST-d1, CLSI-d2, and EUCAST-d2 were 1 (0.125 to 16), 1 (0.125 to 2), and 4 (0.5 to >16), respectively, across all isolates. The similar values for comparator drugs were as follows: posaconazole, 0.25 (≤0.03 to >16), 0.25 (0.06 to >16), and 1 (0.06 to >16); amphotericin, 0.06 (≤0.03 to 0.5), 0.06 (≤0.03 to 0.25), and 0.125 (≤0.03 to 1); voriconazole, 16 (2 to >16), 8 (1 to >16), and >16 (8 to >16), respectively. Isavuconazole activity varied by species:
Lichtheimia corymbifera
, 1 (0.5 to 2), 1 (1 to 2), and 2 (1 to 4);
Lichtheimia ramosa
, 0.25 (0.125 to 0.5), 1 (0.5 to 2), and 2 (0.5 to 4);
Rhizomucor pusillus
, 0.5 (0.5 to 1), 1 (0.125 to 1), and 2 (1 to 2);
Rhizopus microsporus
, 1 (0.5 to 4), 0.5 (0.125 to 1), and 4 (1 to 8); and
Rhizopus oryzae
, 1 (0.5 to 4), 1 (0.125 to 2), and 4 (0.5 to 8), respectively, were more susceptible than
Mucor circinelloides
: group I, 8 (4 to 8), 4 (2 to 4), and 16 (2 to 16), respectively, and group II, 8 (1 to 16), 8 (1 to 8), and 16 (4 to >16), respectively. This was also observed for posaconazole. The essential agreement was best between EUCAST-d1 and CLSI-d2 (75% to 83%). Isavuconazole displayed
in vitro
activity against
Mucorales
isolates with the exception of
Mucor circinelloides
. The MICs were in general 1 to 3 steps higher than those for posaconazole. However, in the clinical setting this may be compensated for by the higher exposure at standard dosing.
Pathophysiological Changes Induced by Pseudomonas aeruginosa Infection Are Involved in MMP-12 and MMP-13 Upregulation in Human Carcinoma Epithelial Cells and a Pneumonia Mouse Model
Authors: Park Ji-Won , Shin In-Sik , Ha Un-Hwan , Oh Sei-Ryang , Kim Jae-Hong , Ahn Kyung-Seop
Published: October 2015

Pseudomonas aeruginosa
infections persist in patients with cystic fibrosis (CF) and drive lung disease progression.
P. aeruginosa
potently activates the innate immune system mostly through the recognition of pathogen-associated molecular patterns, such as flagellin. Matrix metalloproteinases 12 and 13 (MMP-12 and MMP-13, respectively) exacerbate chronic lung infection and inflammation by promoting uncontrolled tissue rearrangements and fibrosis, yet the underlying molecular mechanisms by which this occurs remain largely unknown. In this study, we used quantitative bacteriology, histological examination, and proinflammatory cytokine levels to evaluate the effects of MMP-12 and MMP-13 on
P. aeruginosa
strain K-induced infection and pneumonia in H292 epithelial cells and mice, respectively. Under inflammatory stimulation, mRNA and protein expression levels of proinflammatory mediators were higher in strain K-infected mice and cells than in uninfected counterparts, in which MMP-12 and MMP-13 expression reached levels similar to those observed in epithelial cells. Moreover, we also found that the NF-κB pathway might be involved in the induction of cytokines in response to strain K infection. Taken together, these data suggest that MMP-12 and MMP-13 alter strain K infection in mice and play a role in inflammatory regulation by modulating cytokine levels.
Pancreatic Amylase Is an Environmental Signal for Regulation of Biofilm Formation and Host Interaction in Campylobacter jejuni
Authors: Jowiya Waheed , Brunner Katja , Abouelhadid Sherif , Hussain Haitham A. , Nair Sean P. , Sadiq Sohaib , Williams Lisa K. , Trantham Emma K. , Stephenson Holly , Wren Brendan W. , Bajaj-Elliott Mona , Cogan Tristan A. , Laws Andrew P. , Wade Jim , Dorrell Nick , Allan Elaine
Published: October 2015

Campylobacter jejuni
is a commensal bacterium in the intestines of animals and birds and a major cause of food-borne gastroenteritis in humans worldwide. Here we show that exposure to pancreatic amylase leads to secretion of an α-dextran by
C. jejuni
and that a secreted protease, Cj0511, is required. Exposure of
C. jejuni
to pancreatic amylase promotes biofilm formation
in vitro
, increases interaction with human epithelial cell lines, increases virulence in the
Galleria mellonella
infection model, and promotes colonization of the chicken ileum. We also show that exposure to pancreatic amylase protects
C. jejuni
from stress conditions
in vitro
, suggesting that the induced α-dextran may be important during transmission between hosts. This is the first evidence that pancreatic amylase functions as an interkingdom signal in an enteric microorganism.
A λ Cro-Like Repressor Is Essential for the Induction of Conjugative Transfer of SXT/R391 Elements in Response to DNA Damage
Authors: Poulin-Laprade Dominic , Burrus Vincent
Published: October 2015

Integrative and conjugative elements (ICEs) of the SXT/R391 family are the main contributors to acquired multidrug resistance in the seventh pandemic lineage of
Vibrio cholerae
, the etiological agent of the diarrheal disease cholera. Conjugative transfer of SXT/R391 ICEs is triggered by antibiotics and agents promoting DNA damage through RecA-dependent autoproteolysis of SetR, an ICE-encoded λ CI-like repressor. Here, we describe the role of CroS, a distant λ Cro homolog, as a key component contributing to the regulation of expression of the activator SetCD that orchestrates the expression of the conjugative transfer genes. We show that deletion of
croS
abolishes the SOS response-dependent induction of SXT despite the presence of a functional
setR
gene. Using quantitative reverse transcription-PCR and
lacZ
reporter assays, we also show that CroS represses
setR
and
setCD
expression by binding to operator sites shared with SetR. Furthermore, we provide evidence of an additional operator site bound by SetR and CroS. Finally, we show that SetCD expression generates a positive feedback loop due to SXT excision and replication in a fraction of the cell population. Together, these results refine our understanding of the genetic regulation governing the propagation of major vectors of multidrug resistance.

IMPORTANCE
Healthcare systems worldwide are challenged by an alarming drug resistance crisis caused by the massive and rapid propagation of antibiotic resistance genes and the associated emergence of multidrug-resistant pathogenic bacteria. SXT/R391 ICEs contribute to this phenomenon not only in clinical and environmental vibrios but also in several members of the family
Enterobacteriaceae
. We have identified and characterized here the regulator CroS as a key factor in the stimulation of conjugative transfer of these ICEs in response to DNA-damaging agents. We have also untangled conflicting evidence regarding autoactivation of transfer by the master activator of SXT/R391 ICEs, SetCD. Discovery of CroS provides a clearer and more complete understanding of the regulatory network that governs the dissemination of SXT/R391 ICEs in bacterial populations.
Heparin and hormonal regulation of mRNA synthesis and abundance of autocrine growth factors: relevance to clonal growth of tumors.
Authors: Zvibel I , Halay E , Reid L M
Published: October 2015

Highly sulfated, heparinlike species of heparan sulfate proteoglycans, with heparinlike glycosaminoglycan chains, are extracellular matrix components that are plasma membrane bound in growth-arrested liver cells. Heparins were found to inhibit the growth and lower the clonal growth efficiency of HepG2, a minimally deviant, human hepatoma cell line. Heparan sulfates, closely related glycosaminoglycans present in the extracellular matrix around growing liver cells, had no effect on the growth rate or clonal growth efficiency of HepG2 cells. Neither heparins nor heparan sulfates had any effect on the growth rate or clonal growth efficiency of two poorly differentiated, highly metastatic hepatoma cell lines, SK-Hep-1 and PLC/PRF/5. Heparin's inhibition of growth of HepG2 cells correlated with changes in the mRNA synthesis and abundance of insulinlike growth factor II (IGF II) and transforming growth factor beta (TGF beta). HepG2 cells expressed high basal levels of mRNAs encoding IGF II and TGF beta that were inducible, through transcriptional and posttranscriptional mechanisms, to higher levels by specific heparin-hormone combinations. For both IGF II and TGF beta, the regulation was multifactorial. Transcriptionally, IGF II was regulated by the additive effects of insulin, glucagon, and growth hormone in combination with heparin; TGF beta was regulated primarily by the synergistic effects of insulin and growth hormone in combination with heparin. Posttranscriptionally, the mRNA abundance of the IGF II 4.5- and 3.7-kb transcripts was affected by insulin. Heparin induction of all IGF II transcripts was also dependent on triiodotyronine and prolactin, but it is unknown whether their induction by heparin was via transcriptional or posttranscriptional mechanisms. Heparin-insulin combinations regulated TGF beta posttranscriptionally. The poorly differentiated hepatoma cell lines PLC/PRF/5 and SK-Hep-1 either did not express or constitutively expressed low basal levels of IGF I, IGF II, and TGF beta, whose mRNA synthesis and abundance showed no response to any heparin-hormone combination. We discuss the data as evidence that matrix chemistry is a variable determining the expression of autocrine growth factor genes and the biological responses to them.
Macronuclei and micronuclei in Tetrahymena thermophila contain high-mobility-group-like chromosomal proteins containing a highly conserved eleven-amino-acid putative DNA-binding sequence.
Authors: Schulman I G , Wang T , Wu M , Bowen J , Cook R G , Gorovsky M A , Allis C D
Published: October 2015

HMG (high-mobility-group protein) B and HMG C are abundant nonhistone chromosomal proteins isolated from Tetrahymena thermophila macronuclei with solubilities, molecular weights, and amino acid compositions like those of vertebrate HMG proteins. Genomic clones encoding each of these proteins have been sequenced. Both are single-copy genes that encode single polyadenylated messages whose amounts are 10 to 15 times greater in growing cells than in starved, nongrowing cells. The derived amino acid sequences of HMG B and HMG C contain a highly conserved sequence, the HMG 1 box, found in vertebrate HMGs 1 and 2, and we speculate that this sequence may represent a novel, previously unrecognized DNA-binding motif in this class of chromosomal proteins. Like HMGs 1 and 2, HMGs B and C contain a high percentage of aromatic amino acids. However, the Tetrahymena HMGs are small, are associated with nucleosome core particles, and can be specifically extracted from macronuclei by elutive intercalation, properties associated with vertebrate HMGs 14 and 17, not HMGs 1 and 2. Thus, it appears that these Tetrahymena proteins have features in common with both of the major subgroups of higher eucaryotic HMG proteins. Surprisingly, a linker histone found exclusively in transcriptionally inactive micronuclei also has several HMG-like characteristics, including the ability to be specifically extracted from nuclei by elutive intercalation and the presence of the HMG 1 box. This finding suggests that at least in T. thermophila, proteins with HMG-like properties are not restricted to regions of transcriptionally active chromatin.
Cell cycle-dependent variations in deoxyribonucleotide metabolism among Chinese hamster cell lines bearing the Thy- mutator phenotype.
Authors: Mun B J , Mathews C K
Published: October 2015

Deoxyribonucleotide pool imbalances are frequently mutagenic. We have studied two Chinese hamster ovary cell lines, Thy- 49 and Thy- 303, that were originally characterized by M. Meuth (Mol. Cell. Biol. 1:652-660, 1981). In comparison with wild-type CHO cells, both lines have elevated dCTP/dTTP ratios, resulting from loss of feedback control of CTP synthetase. While asynchronous cultures of both cell lines contain nearly identical deoxyribonucleoside triphosphate (dNTP) pools and both display elevated spontaneous mutation frequencies, the mutation frequencies between the two cell lines differ by as much as 10-fold. We asked whether differences in dNTP pools could be seen in extracts of rapidly isolated nuclei. Small differences, probably not large enough to account for the differences in mutation frequencies, were seen. However, when synchronized S-phase-enriched cell populations were examined, substantial differences were seen, both in whole-cell extracts and in nuclear extracts. Thy- 303 cells, which have higher mutation frequencies than do Thy- 49 cells, also showed the more aberrant dNTP pools. These data indicate that the Thy- 303 line contains a second mutation in addition to the mutation affecting CTP synthetase control. Evidence suggests that this putative second mutation affects an allosteric regulatory site of ribonucleotide reductase. The data on intranuclear dNTP pools in synchronized S-phase cells indicate that higher proportions of cellular dATP and dGTP are found in the nucleus than are corresponding amounts of dCTP and dGTP. Thus, despite the porous nature of the nuclear membrane, there are conditions under which the distributions of deoxyribonucleotides across this membrane are not random.
SDC25, a CDC25-like gene which contains a RAS-activating domain and is a dispensable gene of Saccharomyces cerevisiae.
Authors: Damak F , Boy-Marcotte E , Le-Roscouet D , Guilbaud R , Jacquet M
Published: October 2015

In the yeast Saccharomyces cerevisiae, the CDC25 gene product activates adenylate cyclase through RAS1 and RAS2 gene products. We have recently described the cloning of a DNA fragment which suppresses the cdc25 mutation but not ras1, ras2, or cdc35 mutations. This fragment contains a 5'-truncated open reading frame which shares 47% identity with the C-terminal part of the CDC25 gene. We named the entire gene SDC25. In this paper, we report the cloning, sequencing, and characterization of the complete SDC25 gene. The SDC25 gene is located on the chromosome XII close to the centromere. It is transcribed into a 4-kb-long mRNA that contains an open reading frame of 1,251 codons. Homology with the CDC25 gene extends in the N-terminal part, although the degree of similarity is lower than in the C-terminal part. In contrast with the C-terminal part, the complete SDC25 gene was found not to suppress the CDC25 gene defect. A deletion in the N-terminal part restored the suppressing activity, a result which suggests the existence of a regulatory domain. The SDC25 gene was found to be dispensable for cell growth under usual conditions. No noticeable phenotype was found in the deleted strain.
A chicken beta-actin gene can complement a disruption of the Saccharomyces cerevisiae ACT1 gene.
Authors: Karlsson R , Aspenström P , Byström A S
Published: October 2015

Recently it was demonstrated that beta-actin can be produced in Saccharomyces cerevisiae by using the expression plasmid pY beta actin (R. Karlsson, Gene 68:249-258, 1988), and several site-specific mutants are now being produced in a protein engineering study. To establish a system with which recombinant actin mutants can be tested in vivo and thus enable a correlation to be made with functional effects observed in vitro, a yeast strain lacking endogenous yeast actin and expressing exclusively beta-actin was constructed. This strain is viable but has an altered morphology and a slow-growth phenotype and is temperature sensitive to the point of lethality at 37 degrees C.
Determinants for glycophospholipid anchoring of the Saccharomyces cerevisiae GAS1 protein to the plasma membrane.
Authors: Nuoffer C , Jenö P , Conzelmann A , Riezman H
Published: October 2015

A 125-kDa glycoprotein exposed on the surface of Saccharomyces cerevisiae cells belongs to a class of eucaryotic membrane proteins anchored to the lipid bilayer by covalent linkage to an inositol-containing glycophospholipid. We have cloned the gene (GAS1) encoding the 125-kDa protein (Gas1p) and found that the function of Gas1p is not essential for cell viability. The nucleotide sequence of GAS1 predicts a 60-kDa polypeptide with a cleavable N-terminal signal sequence, potential sites for N- and O-linked glycosylation, and a C-terminal hydrophobic domain. Determination of the anchor attachment site revealed that the C-terminal hydrophobic domain of Gas1p is removed during anchor addition. However, this domain is essential for addition of the glycophospholipid anchor, since a truncated form of the protein failed to become attached to the membrane. Anchor addition was also abolished by a point mutation affecting the hydrophobic character of the C-terminal sequence. We conclude that glycophospholipid anchoring of Gas1p depends on the integrity of the C-terminal hydrophobic domain that is removed during anchor attachment.
Regulation of heat shock factor in Schizosaccharomyces pombe more closely resembles regulation in mammals than in Saccharomyces cerevisiae.
Authors: Gallo G J , Schuetz T J , Kingston R E
Published: October 2015

The heat shock response appears to be universal. All eucaryotes studied encode a protein, heat shock factor (HSF), that is believed to regulate transcription of heat shock genes. This protein binds to a regulatory sequence, the heat shock element, that is absolutely conserved among eucaryotes. We report here the identification of HSF in the fission yeast Schizosaccharomyces pombe. HSF binding was not observed in extracts from normally growing S. pombe (28 degrees C) but was detected in increasing amounts as the temperature of heat shock increased between 39 and 45 degrees C. This regulation is in contrast to that observed in Saccharomyces cerevisiae, in which HSF binding is detectable at both normal and heat shock temperatures. The S. pombe factor bound specifically to the heat shock element, as judged by methylation interference and DNase I protection analysis. The induction of S. pombe HSF was not inhibited by cycloheximide, suggesting that induction occurs posttranslationally, and the induced factor was shown to be phosphorylated. S. pombe HSF was purified to near homogeneity and was shown to have an apparent mobility of approximately 108 kDa. Since heat-induced DNA binding by HSF had previously been demonstrated only in metazoans, the conservation of heat-induced DNA binding by HSF among S. pombe and metazoans suggests that this mode of regulation is evolutionarily ancient.
The strong ADH1 promoter stimulates mitotic and meiotic recombination at the ADE6 gene of Schizosaccharomyces pombe.
Authors: Grimm C , Schaer P , Munz P , Kohli J
Published: October 2015

The effect of the strong promoter from the alcohol dehydrogenase gene on mitotic and meiotic intragenic recombination has been studied at the ade6 locus of the fission yeast Schizosaccharomyces pombe. A 700-bp fragment containing the functional adh1 promoter was used to replace the weak wild-type promoter of the ade6 gene. Analysis of mRNA showed that strains with this ade6::adh1 fusion construct had strongly elevated ade6-specific mRNA levels during vegetative growth as well as in meiosis. These increased levels of mRNA correlated with a 20- to 25-fold stimulation of intragenic recombination in meiosis and a 7-fold increased prototroph formation during vegetative growth. Analysis of flanking marker configurations of prototrophic recombinants indicated that simple conversions as well as conversions associated with crossing over were stimulated in meiosis. The strongest stimulation of recombination was observed when the adh1 promoter was homozygous. Studies with heterologous promoter configurations revealed that the highly transcribed allele was the preferred acceptor of genetic information. The effect of the recombinational hot spot mutation ade6-M26 was also investigated in this system. Its effect was only partly additive to the elevated recombination rate generated by the ade6::adh1 fusion construct.
Characterization of a short, cis-acting DNA sequence which conveys cell cycle stage-dependent transcription in Saccharomyces cerevisiae.
Authors: McIntosh E M , Atkinson T , Storms R K , Smith M
Published: October 2015

Comparison of the 5'-flanking regions of several cell cycle-regulated DNA replication genes of Saccharomyces cerevisiae has revealed the presence of a common sequence, 5'-ACGCGT-3', which is upstream and proximal to mapped transcription initiation sites. This sequence, which is the cleavage site for the restriction endonuclease MluI, is present twice in the upstream region of the yeast thymidylate synthase gene TMP1. Previous studies have implicated these MluI sites as critical components in the cell cycle-dependent transcription of TMP1. In this study, we examined more closely the importance of the ACGCGT sequences for the transcription of this gene. Using site-directed mutagenesis in combination with deletion analysis and subcloning experiments, we found that (i) while both of the TMP1 MluI sites contribute to the total transcription of this gene, the distal site is predominant and (ii) the 9-bp sequence ACGCGTTAA encompassing the distal MluI site exhibits properties of a cell cycle-stage dependent upstream activation sequence element. The results of this study support the notion that the ACGCGT sequence is an integral component of a transcription system which coordinates the cell cycle-dependent expression of DNA replication genes in S. cerevisiae.
Transient activity assays of the Trypanosoma brucei variant surface glycoprotein gene promoter: control of gene expression at the posttranscriptional level.
Authors: Jefferies D , Tebabi P , Pays E
Published: October 2015

The putative promoter of the variant surface glycoprotein (VSG) gene of Trypanosoma brucei was cloned into a plasmid containing the chloramphenicol acetyltransferase (CAT) gene. After electroporation into trypanosomes, this construct directed the expression of the CAT reporter gene. The essential region for promoter activity was found to reside within 88 bp upstream of the putative transcription start site. Transcription of the CAT construct occurred at approximately the same level in both bloodstream and procyclic forms and was resistant to alpha-amanitin. However, CAT expression appeared to be modulated in the two forms of the parasite. Sequences 3' to the gene seemed to be important in this respect, as CAT activity in bloodstream forms was readily detectable only when the 3' region of a VSG cDNA was placed downstream of the CAT gene. Two separate VSG gene promoter sequences, both cloned from T. brucei AnTat 1.3A, were equally able to direct CAT expression, which suggests that there are a number of potential VSG gene promoters in the genome, although usually only one expression site is fully active at any one time.
Intramitochondrial functions regulate nonmitochondrial citrate synthase (CIT2) expression in Saccharomyces cerevisiae.
Authors: Liao X S , Small W C , Srere P A , Butow R A
Published: October 2015

We have examined the effects of perturbation of mitochondrial function on expression of two nuclear genes encoding the mitochondrial and peroxisomal forms of citrate synthase in Saccharomyces cerevisiae, CIT1 and CIT2. CIT2 expression was as much as 30-fold higher in [rho0] petites, than in isochromosomal [rho+] cells, whereas CIT1 expression was slightly down regulated in [rho0] cells. CIT2 expression was also increased in [rho+] cells by inhibition of respiration with antimycin A or in [rho+] cells containing a disruption of the CIT1 gene. These effects were additive, and together they approached the level of CIT2 expression seen in [rho0] cells. Experiments using heterologous gene fusions showed that all of the effects leading to increased expression of CIT2 were transcriptionally controlled through 5'-flanking CIT2 DNA sequences. Analysis of [rho+] and [rho0] cells containing disruptions of CIT1 and CIT2, singly and in combination, showed that the peroxisomal citrate synthase could partially spare the mitochondrial isoform for growth yield in [rho+] but not in [rho0] cells. These studies suggest a physiological role for increased expression of CIT2 in cells with altered mitochondrial function. They also provide additional evidence for a retrograde path of communication from mitochondria to the nucleus in yeast cells.
In vivo pre-tRNA processing in Saccharomyces cerevisiae.
Authors: O'Connor J P , Peebles C L
Published: October 2015

We have surveyed intron-containing RNAs of the yeast Saccharomyces cerevisiae by filter hybridization with pre-tRNA intron-specific oligonucleotide probes. We have classified various RNAs as pre-tRNAs, splicing intermediates, or excised intron products according to apparent size and structure. Linear, excised intron products were detected, and one example was isolated and sequenced directly. Additional probes designed to detect other precursor sequences were used to verify the identification of several intermediates. Pre-tRNA species with both 5' leader and 3' extension, with 3' extension only, and with mature ends were distinguished. From these results, we conclude that the processing reactions used to remove the 5' leader and 3' extension from the transcript are ordered 5' end trimming before 3' end trimming. Splicing intermediates containing the 5' exon plus the intron were detected. The splice site cleavage reactions are probably ordered 3' splice site cleavage before 5' splice site cleavage. Surprisingly, we also detected a splicing intermediate with the 5' leader and a spliced product with both 5' leader and 3' extension. Evidently, splicing and end trimming are not ordered relative to each other, splicing occurring either before or after end trimming.
Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system.
Authors: Jessberger R , Berg P
Published: October 2015

We have designed an in vitro system using mammalian nuclear extracts, or fractions derived from them, that can restore the sequences missing at double-strand breaks (gaps) or in deletions. The recombination substrates consist of (i) recipient DNA, pSV2neo with gaps or deletions ranging from 70 to 390 bp in the neo sequence, and (ii) donor DNAs with either complete homology to the recipient (pSV2neo) or plasmids whose homology with pSV2neo is limited to a 1.0- to 1.3-kbp neo segment spanning the gaps or deletions. Incubation of these substrates with various enzyme fractions results in repair of the recipient DNA's disrupted neo gene. The recombinational repair was monitored by transforming recA Escherichia coli to kanamycin resistance and by a new assay which measures the extent of DNA strand transfer from the donor substrate to the recipient DNA. Thus, either streptavidin- or antidigoxigenin-tagged beads are used to separate the biotinylated or digoxigeninylated recipient DNA, respectively, after incubation with the isotopically labeled donor DNA. In contrast to the transfection assay, the DNA strand transfer measurements are direct, quantitative, rapid, and easy, and they provide starting material for the characterization of the recombination products and intermediates. Accordingly, DNA bound to beads serves as a suitable template for the polymerase chain reaction. With appropriate pairs of oligonucleotide primers, we have confirmed that both gaps and deletions are fully repaired, that deletions can be transferred from the recipient DNA to the donor's intact neo sequence, and that cointegrant molecules containing donor and recipient DNA sequences are formed.
Dictyostelium discoideum lipids modulate cell-cell cohesion and cyclic AMP signaling.
Authors: Fontana D R , Luo C S , Phillips J C
Published: October 2015

During Dictyostelium discoideum development, cell-cell communication is mediated through cyclic AMP (cAMP)-induced cAMP synthesis and secretion (cAMP signaling) and cell-cell contact. Cell-cell contact elicits cAMP secretion and modulates the magnitude of a subsequent cAMP signaling response (D. R. Fontana and P. L. Price, Differentiation 41:184-192, 1989), demonstrating that cell-cell contact and cAMP signaling are not independent events. To identify components involved in the contact-mediated modulation of cAMP signaling, amoebal membranes were added to aggregation-competent amoebae in suspension. The membranes from aggregation-competent amoebae inhibited cAMP signaling at all concentrations tested, while the membranes from vegetative amoebae exhibited a concentration-dependent enhancement or inhibition of cAMP signaling. Membrane lipids inhibited cAMP signaling at all concentrations tested. The lipids abolished cAMP signaling by blocking cAMP-induced adenylyl cyclase activation. The membrane lipids also inhibited amoeba-amoeba cohesion at concentrations comparable to those which inhibited cAMP signaling. The phospholipids and neutral lipids decreased cohesion and inhibited the cAMP signaling response. The glycolipid/sulfolipid fraction enhanced cohesion and cAMP signaling. Caffeine, a known inhibitor of cAMP-induced adenylyl cyclase activation, inhibited amoeba-amoeba cohesion. These studies demonstrate that endogenous lipids are capable of modulating amoeba-amoeba cohesion and cAMP-induced activation of the adenylyl cyclase. These results suggest that cohesion may modulate cAMP-induced adenylyl cyclase activation. Because the complete elimination of cohesion is accompanied by the complete elimination of cAMP signaling, these results further suggest that cohesion may be necessary for cAMP-induced adenylyl cyclase activation in D. discoideum.
Sgs-3 chromatin structure and trans-activators: developmental and ecdysone induction of a glue enhancer-binding factor, GEBF-I, in Drosophila larvae.
Authors: Georgel P , Ramain P , Giangrande A , Dretzen G , Richards G , Bellard M
Published: October 2015

The transcription of the Drosophila melanogaster 68C salivary gland glue gene Sgs-3 involves the interaction of a distal and a proximal regulatory region. These are marked in vivo by a specific chromatin structure which is established sequentially during development, starting early in embryogenesis. The distal region is characterized by a stage- and tissue-specific DNase I hypersensitive site. A stage- and tissue-specific factor, GEBF-I, binds in this region and is missing in 2B5 mutant larvae which lack Sgs-3 transcripts. This binding involves the simultaneous interaction with two distinct DNA sequences which induces conformational changes in the protein. Salivary glands acquire competence to respond to ecdysone in the mid-third larval instar, whereafter the hormone rapidly induces both the GEBF-I protein and Sgs-3 transcription.
Alternative initiation of translation determines cytoplasmic or nuclear localization of basic fibroblast growth factor.
Authors: Bugler B , Amalric F , Prats H
Published: October 2015

Three forms of basic fibroblast growth factor (bFGF), initiated at an AUG (18 kDa) and two CUG (21 and 22.5 kDa) start codons, were produced following transfection of COS cells with human hepatoma bFGF cDNA. The subcellular localization of the different forms was investigated directly or by using chimeric genes constructed by fusion of the bFGF and chloramphenicol acetyltransferase open reading frames. The AUG-initiated proteins were cytoplasmic, while the CUG-initiated forms were nuclear. The signal sequence responsible for the nuclear localization of bFGF is contained within 37 amino acid residues between the second CUG and the AUG start codons. Alternative initiation of translation regulates the subcellular localization of bFGF and thus could modulate its role in cell growth and differentiation control.
Induction of growth arrest by a temperature-sensitive p53 mutant is correlated with increased nuclear localization and decreased stability of the protein.
Authors: Ginsberg D , Michael-Michalovitz D , Ginsberg D , Oren M
Published: October 2015

A temperature-sensitive mutant of p53, p53Val-135, was found to be able to arrest cell proliferation when overexpressed at 32.5 degrees C. While much of the protein was cytoplasmic in cells proliferating at 37.5 degrees C, it became predominantly nuclear at 32.5 degrees C. Concomitantly, p53Val-135 became destabilized, although not to the extent seen in primary fibroblasts.