bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of Borate Bioactive Glass and Calcium Sulfate as Implants for the Local Delivery of Teicoplanin in the Treatment of Methicillin-Resistant Staphylococcus aureus-Induced Osteomyelitis in a Rabbit Model
Authors: Jia Wei-Tao , Fu Qiang , Huang Wen-Hai , Zhang Chang-Qing , Rahaman Mohamed N.
Published: September 2015

There is growing interest in biomaterials that can cure bone infection and also regenerate bone. In this study, two groups of implants composed of 10% (wt/wt) teicoplanin (TEC)-loaded borate bioactive glass (designated TBG) or calcium sulfate (TCS) were created and evaluated for their ability to release TEC
in vitro
and to cure methicillin-resistant
Staphylococcus aureus
(MRSA)-induced osteomyelitis in a rabbit model. When immersed in phosphate-buffered saline (PBS), both groups of implants provided a sustained release of TEC at a therapeutic level for up to 3 to 4 weeks while they were gradually degraded and converted to hydroxyapatite. The TBG implants showed a longer duration of TEC release and better retention of strength as a function of immersion time in PBS. Infected rabbit tibiae were treated by debridement, followed by implantation of TBG or TCS pellets or intravenous injection with TEC, or were left untreated. Evaluation at 6 weeks postimplantation showed that the animals implanted with TBG or TCS pellets had significantly lower radiological and histological scores, lower rates of MRSA-positive cultures, and lower bacterial loads than those preoperatively and those of animals treated intravenously. The level of bone regeneration was also higher in the defects treated with the TBG pellets. The results showed that local TEC delivery was more effective than intravenous administration for the treatment of MRSA-induced osteomyelitis. Borate glass has the advantages of better mechanical strength, more desirable kinetics of release of TEC, and a higher osteogenic capacity and thus could be an effective alternative to calcium sulfate for local delivery of TEC.
K13-Propeller Polymorphisms in Plasmodium falciparum Isolates from Patients in Mayotte in 2013 and 2014
Authors: Torrentino-Madamet Marylin , Collet Louis , Lepère Jean François , Benoit Nicolas , Amalvict Rémy , Ménard Didier , Pradines Bruno
Published: September 2015

Plasmodium falciparum
isolates were collected from 29 malaria patients treated with artemether-lumefantrine in Mayotte in 2013 and 2014. Twenty-four cases (83%) consisted of imported malaria. Seventeen percent of the isolates presented mutations in one of the six K13-propeller blades (N490H, F495L, N554H/K, and E596G). A total of 23.8% of the isolates from the Union of Comoros showed K13-propeller polymorphisms. Three of the 18 isolates (16.7%) from Grande Comore showed polymorphisms (N490H, N554K, and E596G).
Different Therapeutic Outcomes of Benznidazole and VNI Treatments in Different Genders in Mouse Experimental Models of Trypanosoma cruzi Infection
Authors: Guedes-da-Silva F. H. , Batista D. G. J. , da Silva C. F. , Meuser M. B. , Simões-Silva M. R. , de Araújo J. S. , Ferreira C. G. , Moreira O. C. , Britto C. , Lepesheva G. I. , Soeiro Maria de Nazaré C.
Published: September 2015

The lack of translation between preclinical assays and clinical trials for novel therapies for Chagas disease (CD) indicates a need for more feasible and standardized protocols and experimental models. Here, we investigated the effects of treatment with benznidazole (Bz) and with the potent experimental
T. cruzi
CYP51 inhibitor VNI in mouse models of Chagas disease by using different animal genders and parasite strains and employing distinct types of therapeutic schemes. Our findings confirm that female mice are less vulnerable to the infection than males, show that male models are less susceptible to treatment with both Bz and VNI, and thus suggest that male models are much more suitable for selection of the most promising antichagasic agents. Additionally, we have found that preventive protocols (compound given at 1 dpi) result in higher treatment success rates, which also should be avoided during advanced steps of
in vivo
trials of novel anti-
T. cruzi
drug candidates. Another consideration is the relevance of immunosuppression methods in order to verify the therapeutic profile of novel compounds, besides the usefulness of molecular diagnostic tools (quantitative PCR) to ascertain compound efficacy in experimental animals. Our study aims to contribute to the development of more reliable methods and decision gates for
in vivo
assays of novel antiparasitic compounds in order to move them from preclinical to clinical trials for CD.
Impact of Hydroxychloroquine-Loaded Polyurethane Intravaginal Rings on Lactobacilli
Authors: Traore Yannick Leandre , Chen Yufei , Bernier Anne-Marie , Ho Emmanuel A.
Published: September 2015

The use of polymeric devices for controlled sustained delivery of drugs is a promising approach for the prevention of HIV-1 infection. Unfortunately, certain microbicides, when topically applied vaginally, may be cytotoxic to vaginal epithelial cells and the protective microflora present within the female genital tract. In this study, we evaluated the impact of hydroxychloroquine (HCQ)-loaded, reservoir-type, polyurethane intravaginal rings (IVRs) on the growth of
Lactobacillus crispatus
and
Lactobacillus jensenii
and on the viability of vaginal and ectocervical epithelial cells. The IVRs were fabricated using hot-melt injection molding and were capable of providing controlled release of HCQ for 24 days, with mean daily release rates of 17.01 ± 3.6 μg/ml in sodium acetate buffer (pH 4) and 29.45 ± 4.84 μg/ml in MRS broth (pH 6.2). Drug-free IVRs and the released HCQ had no significant effects on bacterial growth or the viability of vaginal or ectocervical epithelial cells. Furthermore, there was no significant impact on the integrity of vaginal epithelial cell monolayers, in comparison with controls, as measured by transepithelial electrical resistance. Overall, this is the first study to evaluate the effects of HCQ-loaded IVRs on the growth of vaginal flora and the integrity of vaginal epithelial cell monolayers.
In Vitro
Activity of Ceftaroline against Staphylococcus aureus Isolates Collected in 2012 from Latin American Countries as Part of the AWARE Surveillance Program
Authors: Biedenbach Douglas J. , Hoban Daryl J. , Reiszner Edina , Lahiri Sushmita D. , Alm Richard A. , Sahm Daniel F. , Bouchillon Samuel K. , Ambler Jane E.
Published: September 2015

The
in vitro
activities of ceftaroline and comparators, using broth microdilution, were determined against 1,066
Staphylococcus aureus
isolates from hospitalized patients. Seventeen medical centers from Latin American countries contributed isolates. Methicillin-resistant
S. aureus
(MRSA) percentages ranged from 46% (Brazil) to 62% (Argentina). All methicillin-susceptible
S. aureus
(MSSA) isolates were susceptible to ceftaroline. Ceftaroline activity against MRSA varied with MIC
90
s of 0.5 (Venezuela) to 2 (Brazil, Chile, and Colombia) μg/ml, which was the highest MIC value. ST-5 was the most common sequence type.
The Pathogen-Derived Aminoglycoside Resistance 16S rRNA Methyltransferase NpmA Possesses Dual m
1
A1408/m
1
G1408 Specificity
Authors: Zelinskaya Natalia , Witek Marta A. , Conn Graeme L.
Published: September 2015

Chemical modification of 16S rRNA can confer exceptionally high-level resistance to a diverse set of aminoglycoside antibiotics. Here, we show that the pathogen-derived enzyme NpmA possesses dual m
1
A1408/m
1
G1408 activity, an unexpected property apparently unique among the known aminoglycoside resistance 16S rRNA (m
1
A1408) methyltransferases. Although the biological significance of this activity remains to be determined, such mechanistic variation in enzymes acquired by pathogens has significant implications for development of inhibitors of these emerging resistance determinants.
In Vitro
Activity of Miltefosine against Candida albicans under Planktonic and Biofilm Growth Conditions and
In Vivo
Efficacy in a Murine Model of Oral Candidiasis
Authors: Vila Taissa Vieira Machado , Chaturvedi Ashok K. , Rozental Sonia , Lopez-Ribot Jose L.
Published: September 2015

The generation of a new antifungal against
Candida albicans
biofilms has become a major priority, since biofilm formation by this opportunistic pathogenic fungus is usually associated with an increased resistance to azole antifungal drugs and treatment failures. Miltefosine is an alkyl phospholipid with promising antifungal activity. Here, we report that, when tested under planktonic conditions, miltefosine displays potent
in vitro
activity against multiple fluconazole-susceptible and -resistant
C. albicans
clinical isolates, including isolates overexpressing efflux pumps and/or with well-characterized Erg11 mutations. Moreover, miltefosine inhibits
C. albican
s biofilm formation and displays activity against preformed biofilms. Serial passage experiments confirmed that miltefosine has a reduced potential to elicit resistance, and screening of a library of
C. albicans
transcription factor mutants provided additional insight into the activity of miltefosine against
C. albicans
growing under planktonic and biofilm conditions. Finally, we demonstrate the
in vivo
efficacy of topical treatment with miltefosine in the murine model of oropharyngeal candidiasis. Overall, our results confirm the potential of miltefosine as a promising antifungal drug candidate, in particular for the treatment of azole-resistant and biofilm-associated superficial candidiasis.
Transcriptome Analysis of Aspergillus flavus Reveals
veA
-Dependent Regulation of Secondary Metabolite Gene Clusters, Including the Novel Aflavarin Cluster
Authors: Cary J. W. , Han Z. , Yin Y. , Lohmar J. M. , Shantappa S. , Harris-Coward P. Y. , Mack B. , Ehrlich K. C. , Wei Q. , Arroyo-Manzanares N. , Uka V. , Vanhaecke L. , Bhatnagar D. , Yu J. , Nierman W. C. , Johns M. A. , Sorensen D. , Shen H. , De Saeger S. , Diana Di Mavungu J. , Calvo A. M.
Published: September 2015

The global regulatory
veA
gene governs development and secondary metabolism in numerous fungal species, including
Aspergillus flavus
. This is especially relevant since
A. flavus
infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are
veA
dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the
A. flavus
genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that
veA
is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of
veA
. One of the clusters under the influence of
veA
is cluster 39. The absence of
veA
results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin.
Interleukin-12-Producing CD103+CD11b−CD8+Dendritic Cells Are Responsible for Eliciting Gut Intraepithelial Lymphocyte Response against Encephalitozoon cuniculi
Authors: Moretto Magali M. , Harrow Danielle I. , Hawley Teresa S. , Khan Imtiaz A.
Published: September 2015

Microsporidia, which belong to the kingdomFungi, are important opportunistic pathogens in HIV-infected populations and organ transplant recipients that are often associated with a broad range of symptoms, such as diarrhea, nephritis, and encephalitis. Natural infection occurs via the oral route, and as a consequence, gut immunity plays an important role in restricting the dissemination of these pathogens. Studies from our laboratory have reported that the pathogens induce a rapid intraepithelial lymphocyte (IEL) response important for host protection. Although mucosal dendritic cells (DC) are likely involved in triggering an antigen-specific IEL response, the specific subset(s) responsible has yet to be identified. Toward this goal, we demonstrate a very important role for mucosal CD11b−CD8+DC in the initiation of an antigen-specific IELin vivo. Effectively, afterEncephalitozoon cuniculiinfection, CD11b−CD8+DC were activated in the lamina propria (LP) and acquired the ability to process retinoic acid (RA). However, this subset did not produce interleukin 12 (IL-12) but upregulated CD103, which is essential for migration to the mesenteric lymph nodes (MLN). Interestingly, CD103+CD11b−CD8+DC in the MLN, in addition to processing RA, also secreted IL-12 and were responsible for gut imprinting specificity on mucosal CD8 T cells. To the best of our knowledge, this is the first report describing the importance of MLN CD103+CD11b−CD8+DC isolated from infected animals in the generation of an IEL response against a live pathogen.
Endovascular Infections Caused by Methicillin-Resistant Staphylococcus aureus Are Linked to Clonal Complex-Specific Alterations in Binding and Invasion Domains of Fibronectin-Binding Protein A as Well as the Occurrence of
fnbB
Authors: Xiong Yan Q. , Sharma-Kuinkel Batu K. , Casillas-Ituarte Nadia N. , Fowler Vance G. , Rude Thomas , DiBartola Alex C. , Lins Roberto D. , Abdel-Hady Wessam , Lower Steven K. , Bayer Arnold S.
Published: September 2015

Endovascular infections caused by
Staphylococcus aureus
involve interactions with fibronectin present as extracellular matrix or surface ligand on host cells. We examined the expression, structure, and binding activity of the two major
S. aureus
fibronectin-binding proteins (FnBPA, FnBPB) in 10 distinct, methicillin-resistant clinical isolates from patients with either persistent or resolving bacteremia. The persistent bacteremia isolates (
n
= 5) formed significantly stronger bonds with immobilized fibronectin as determined by dynamic binding measurements performed with atomic force microscopy. Several notable differences were also observed when the results were grouped by clonal complex 5 (CC5) strains (
n
= 5) versus CC45 strains (
n
= 5). Fibronectin-binding receptors on CC5 formed stronger bonds with immobilized fibronectin (
P
< 0.001). The
fnbA
gene was expressed at higher levels in CC45, whereas
fnbB
was found in only CC5 isolates. The
fnbB
gene was not sequenced because all CC45 isolates lacked this gene. Instead, comparisons were made for
fnbA
, which was present in all 10 isolates. Sequencing of
fnbA
revealed discrete differences within high-affinity, fibronectin-binding repeats (FnBRs) of FnBPA that included (i) 5-amino-acid polymorphisms in FnBR-9, FnBR-10, and FnBR-11 involving charged or polar side chains, (ii) an extra, 38-amino-acid repeat inserted between FnBR-9 and FnBR-10 exclusively seen in CC45 isolates, and (iii) CC5 isolates had the SVDFEED epitope in FnBR-11 (a sequence shown to be essential for fibronectin binding), while this sequence was replaced in all CC45 isolates with GIDFVED (a motif known to favor host cell invasion at the cost of reduced fibronectin binding). These complementary sequence and binding data suggest that differences in
fnbA
and
fnbB
, particularly polymorphisms and duplications in FnBPA, give
S. aureus
two distinct advantages in human endovascular infections: (i) FnBPs similar to that of CC5 enhance ligand binding and foster initiation of disease, and (ii) CC45-like FnBPs promote cell invasion, a key attribute in persistent endovascular infections.
Response of Vibrio cholerae to the Catecholamine Hormones Epinephrine and Norepinephrine
Authors: Halang Petra , Toulouse Charlotte , Geißel Bernadette , Michel Bernd , Flauger Birgit , Müller Manuel , Voegele Ralf T. , Stefanski Volker , Steuber Julia
Published: September 2015

In
Escherichia coli
or
Salmonella enterica
, the stress-associated mammalian hormones epinephrine (E) and norepinephrine (NE) trigger a signaling cascade by interacting with the QseC sensor protein. Here we show that
Vibrio cholerae
, the causative agent of cholera, exhibits a specific response to E and NE. These catecholates (0.1 mM) enhanced the growth and swimming motility of
V. cholerae
strain O395 on soft agar in a medium containing calf serum, which simulated the environment within the host. During growth, the hormones were converted to degradation products, including adrenochrome formed by autooxidation with O
2
or superoxide. In
E. coli
, the QseC sensor kinase, which detects the autoinducer AI-3, also senses E or NE. The genome of
V. cholerae
O395 comprises an open reading frame coding for a putative protein with 29% identity to
E. coli
QseC. Quantitative reverse transcriptase PCR (qRT-PCR) experiments revealed increased transcript levels of the
qseC
-like gene and of
pomB
, a gene encoding a structural component of the flagellar motor complex, under the influence of E or NE. Phentolamine blocks the response of
E. coli
QseC to E or NE. A
V. cholerae
mutant devoid of the
qseC
-like gene retained the phentolamine-sensitive motility in the presence of E, whereas NE-stimulated motility was no longer inhibited by phentolamine. Our study demonstrates that
V. cholerae
senses the stress hormones E and NE. A sensor related to the histidine kinase QseC from
E. coli
is identified and is proposed to participate in the sensing of NE.

IMPORTANCE
Vibrio cholerae
is a Gram-negative bacterium that may cause cholera, a severe illness with high mortality due to acute dehydration caused by diarrhea and vomiting. Pathogenic
V. cholerae
strains possess virulence factors like the cholera toxin (CTX) and the toxin-coregulated pilus (TCP) produced in response to signals provided by the host. In pathogenic enterobacteria, the stress-associated hormones epinephrine (E) and norepinephrine (NE) of the human host act as signal molecules for the production of virulence factors and promote bacterial growth by the sequestration of iron from the host. Here we show that
V. cholerae
, like some enterobacteria, benefits from these stress hormones and possesses a sensor to recognize them.
The Sugar Kinase That Is Necessary for the Catabolism of Rhamnose in Rhizobium leguminosarum Directly Interacts with the ABC Transporter Necessary for Rhamnose Transport
Authors: Rivers Damien M. R. , Oresnik Ivan J.
Published: September 2015

Rhamnose catabolism in
Rhizobium leguminosarum
was found to be necessary for the ability of the organism to compete for nodule occupancy. Characterization of the locus necessary for the catabolism of rhamnose showed that the transport of rhamnose was dependent upon a carbohydrate uptake transporter 2 (CUT2) ABC transporter encoded by
rhaSTPQ
and on the presence of RhaK, a protein known to have sugar kinase activity. A linker-scanning mutagenesis analysis of
rhaK
showed that the kinase and transport activities of RhaK could be separated genetically. More specifically, two pentapeptide insertions defined by the alleles
rhaK72
and
rhaK73
were able to uncouple the transport and kinase activities of RhaK, such that the kinase activity was retained, but cells carrying these alleles did not have measurable rhamnose transport rates. These linker-scanning alleles were localized to the C terminus and N terminus of RhaK, respectively. Taken together, the data led to the hypothesis that RhaK might interact either directly or indirectly with the ABC transporter defined by
rhaSTPQ
. In this work, we show that both N- and C-terminal fragments of RhaK are capable of interacting with the N-terminal fragment of the ABC protein RhaT using a 2-hybrid system. Moreover, if RhaK fragments carrying either the
rhaK72
or
rhaK73
allele were used, this interaction was abolished. Phylogenetic and bioinformatic analysis of the RhaK fragments suggested that a conserved region in the N terminus of RhaK may represent a putative binding domain. Alanine-scanning mutagenesis of this region followed by 2-hybrid analysis revealed that a substitution of any of the conserved residues greatly affected the interaction between RhaT and RhaK fragments, suggesting that the sugar kinase RhaK and the ABC protein RhaT interact directly.

IMPORTANCE
ABC transporters involved in the transport of carbohydrates help define the overall physiological fitness of bacteria. The two largest groups of transporters are the
c
arbohydrate
u
ptake
t
ransporter classes 1 and 2 (CUT1 and CUT2, respectively). This work provides the first evidence that a kinase that is necessary for the catabolism of a sugar can directly interact with a domain from the ABC protein that is necessary for its transport.
In Silico
Discovery and
In Vitro
Validation of Catechol-Containing Sulfonohydrazide Compounds as Potent Inhibitors of the Diguanylate Cyclase PleD
Authors: Fernicola Silvia , Paiardini Alessandro , Giardina Giorgio , Rampioni Giordano , Leoni Livia , Cutruzzolà Francesca , Rinaldo Serena
Published: September 2015

Biofilm formation is responsible for increased antibiotic tolerance in pathogenic bacteria. Cyclic di-GMP (c-di-GMP) is a widely used second-messenger signal that plays a key role in bacterial biofilm formation. c-di-GMP is synthesized by diguanylate cyclases (DGCs), a conserved class of enzymes absent in mammals and hence considered attractive molecular targets for the development of antibiofilm agents. Here, the results of a virtual screening approach aimed at identifying small-molecule inhibitors of the DGC PleD from
Caulobacter crescentus
are described. A three-dimensional (3D) pharmacophore model, derived from the mode of binding of GTP to the active site of PleD, was exploited to screen the ZINC database of compounds. Seven virtual hits were tested
in vitro
for their ability to inhibit the activity of purified PleD by using circular dichroism spectroscopy. Two drug-like molecules with a catechol moiety and a sulfonohydrazide scaffold were shown to competitively inhibit PleD at the low-micromolar range (50% inhibitory concentration [IC
50
] of ∼11 μM). Their predicted binding mode highlighted key structural features presumably responsible for the efficient inhibition of PleD by both hits. These molecules represent the most potent
in vitro
inhibitors of PleD identified so far and could therefore result in useful leads for the development of novel classes of antimicrobials able to hamper biofilm formation.

IMPORTANCE
Biofilm-mediated infections are difficult to eradicate, posing a threatening health issue worldwide. The capability of bacteria to form biofilms is almost universally stimulated by the second messenger c-di-GMP. This evidence has boosted research in the last decade for the development of new antibiofilm strategies interfering with c-di-GMP metabolism. Here, two potent inhibitors of c-di-GMP synthesis have been identified
in silico
and characterized
in vitro
by using the well-characterized DGC enzyme PleD from
C. crescentus
as a structural template and molecular target. Given that the protein residues implied as crucial for enzyme inhibition are found to be highly conserved among DGCs, the outcome of this study could pave the way for the future development of broad-spectrum antibiofilm compounds.
Recombinationless meiosis in Saccharomyces cerevisiae.
Authors: Malone R E , Esposito R E
Published: September 2015

We have utilized the single equational meiotic division conferred by the spo13-1 mutation of Saccharomyces cerevisiae (S. Klapholtz and R. E. Esposito, Genetics 96:589-611, 1980) as a technique to study the genetic control of meiotic recombination and to analyze the meiotic effects of several radiation-sensitive mutations (rad6-1, rad50-1, and rad52-1) which have been reported to reduce meiotic recombination (Game et al., Genetics 94:51-68, 1980); Prakash et al., Genetics 94:31-50, 1980). The spo13-1 mutation eliminates the meiosis I reductional segregation, but does not significantly affect other meiotic events (including recombination). Because of the unique meiosis it confers, the spo13-1 mutation provides an opportunity to recover viable meiotic products in a Rec- background. In contrast to the single rad50-1 mutant, we found that the double rad50-1 spo13-1 mutant produced viable ascospores after meiosis and sporulation. These spores were nonrecombinant: meiotic crossing-over was reduced at least 150-fold, and no increase in meiotic gene conversion was observed over mitotic background levels. The rad50-1 mutation did not, however, confer a Rec- phenotype in mitosis; rather, it increased both spontaneous crossing-over and gene conversion. The spore inviability conferred by the single rad6-1 and rad52-1 mutations was not eliminated by the presence of the spo13-1 mutation. Thus, only the rad50 gene has been unambiguously identified by analysis of viable meiotic ascospores as a component of the meiotic recombination system.
Elevated levels of (2'-5')oligoadenylic acid polymerase activity in growth-arrested human lymphoblastoid Namalva cells.
Authors: Krishnan I , Baglioni C
Published: September 2015

(2'-5')Oligoadenylic acid [(2'-5')An] polymerase activity was measured in extracts of human lymphoblastoid cells of the Namalva line cultured under different conditions. Exponentially growing cells had a relatively low polymerase activity level, whereas cells grown to limit density showed elevated levels. When fresh medium was added to growth-arrested cells, (2'-5')An polymerase activity decreased concomitantly with the initiation of active deoxyribonucleic acid synthesis. An increase in polymerase activity level was also observed after exponentially growing cells were transferred from medium containing 20% serum to fresh medium containing 0.2% serum. These cells diminished deoxyribonucleic acid synthesis and remained quiescent until 20% serum was again added. Polymerase activity level decreased as the cells entered into S phase. The addition of the inhibitor of deoxyribonucleic acid synthesis, hydroxyurea, to exponentially growing cells did not increase polymerase level, indicating that cells blocked in S phase and at the G1-S boundary maintained the basal level of this enzyme. Degradation of labeled (2'-5')An was measured in extracts of Namalva cells cultured under different conditions, but no significant differences among degradative activities were observed. Since (2'-5')An polymerase activity is one of the enzymatic activities induced by interferon, we measured interferon titers in Namalva cell medium. Less than 1 reference unit per ml was detected in cells grown under different conditions. Moreover, the increase in (2'-5')An polymerase activity level in cells transferred from 20 to 0.2% serum was not prevented by including anti-lymphoblastoid interferon antibody in the medium. These results suggest that the activity level of (2'-5')An polymerase is regulated in Namalva cells on the basis of the growth status of the cells and that this regulatory mechanism is apparently not activated by interferon.
Coordinate control of syntheses of ribosomal ribonucleic acid and ribosomal proteins during nutritional shift-up in Saccharomyces cerevisiae.
Authors: Kief D R , Warner J R
Published: September 2015

We investigated the regulation of ribosome synthesis in Saccharomyces cerevisiae growing at different rates and in response to a growth stimulus. The ribosome content and the rates of synthesis of ribosomal ribonucleic acid and of ribosomal proteins were compared in cultures growing in minimal medium with either glucose or ethanol as a carbon source. The results demonstrated that ribosome content is proportional to growth rate. Moreover, these steady-state concentrations are regulated at the level of synthesis of ribosomal precursor ribonucleic acid and of ribosomal proteins. When cultures growing on ethanol were enriched with glucose, the rate of ribosomal ribonucleic acid synthesis, measured by pulsing cells with [methyl-3H]methionine, increased by 40% within 5 min, doubled within 15 min, and reached a steady state characteristic of the new growth medium by 30 min. Labeling with [3H]leucine reveal a coordinate increase in the rate of synthesis of 30 or more ribosomal proteins as compared with that of total cellular proteins. Their synthesis was stimulated approximately 2.5-fold within 15 min and nearly 4-fold within 60 min. The data suggest that S. cerevisiae responds to a growth stimulus by preferential stimulation of the synthesis of ribosomal ribonucleic acid and ribosomal proteins.
Hierarchy of elements regulating synthesis of ribosomal proteins in Saccharomyces cerevisiae.
Authors: Kief D R , Warner J R
Published: September 2015

Saccharomyces cerevisiae cells respond to a heat shock by temporarily slowing the synthesis of ribosomal proteins (C. Gorenstein and J. R. Warner, Proc. Natl. Acad. Sci. U.S.A. 73:1574-1551, 1976). When cultures growing oxidatively on ethanol as the sole carbon source were shifted from 23 to 36 degrees C, the synthesis of ribosomal proteins was coordinately inhibited twice as rapidly and 45% more severely than in comparable cultures growing fermentatively on glucose. Within 15 min, the relative rates of synthesis of at least 30 ribosomal proteins declined to less than one-sixth their initial values, whereas the overall rate of protein synthesis increased at least threefold. We suggest that this is due primarily to controls at the level of synthesis of messenger ribonucleic acid for ribosomal proteins but may also involve changes in messenger ribonucleic acid stability. In contrast, a nutritional shift-up causes a stimulation of the synthesis of ribosomal proteins. Experiments designed to determine the hierarchy of stimuli affecting the synthesis of these proteins demonstrated that temperature shock was dominant to glucose stimulation. When a culture growing on ethanol was shifted from 23 to 36 degrees C and glucose was added shortly afterward, the decline in ribosomal protein synthesis continued unabated. However, in wild-type cells ribosomal protein synthesis began to recover within 15 min. In mutants temperature sensitive for ribosome synthesis, e.g., rna2, there was no recovery in the synthesis of most ribosomal proteins, suggesting that the product of rna2 is essential for the production of these proteins under all vegetative conditions.
Comparison between the viral transforming gene (src) of recovered avian sarcoma virus and its cellular homolog.
Authors: Takeya T , Hanafusa H , Junghans R P , Ju G , Skalka A M
Published: September 2015

Recovered avian sarcoma viruses are recombinants between transformation-defective mutants of Rous sarcoma virus and the chicken cellular gene homologous to the src gene of Rous sarcoma virus. We have constructed and analyzed molecular clones of viral deoxyribonucleic acid from recovered avian sarcoma virus and its transformation-competent progenitor, the Schmidt-Ruppin A strain of Rous sarcoma virus. A 2.0-megadalton EcoRI fragment containing the entire src gene from each of these clones was subcloned and characterized. These fragments were also used as probes to isolate recombinant phage clones containing the cellular counterpart of the viral src gene, termed cellular src, from a lambda library of chicken deoxyribonucleic acid. The structure of cellular src was analyzed by restriction endonuclease mapping and electron microscopy. Restriction endonuclease mapping revealed extensive similarity between the src regions of Rous sarcoma virus and recovered avian sarcoma virus, but striking differences between the viral src's and cellular src. Electron microscopic analysis of heteroduplexes between recovered virus src and cellular src revealed a 1.8-kilobase region of homology. In the cellular gene, the homologous region was interrupted by seven nonhomologous regions which we interpret to be intervening sequences. We estimate the minimum length of cellular src to be about 7.2 kilobases. These findings have implications concerning the mechanism of formation of recovered virus src and possibly other cell-derived retrovirus transforming genes.
Relationship of amplified dihydrofolate reductase genes to double minute chromosomes in unstably resistant mouse fibroblast cell lines.
Authors: Brown P C , Beverley S M , Schimke R T
Published: September 2015

Murine 3T6 selected in increasing concentrations of methotrexate were unstable with respect to dihydrofolate reductase overproduction and methotrexate resistance when they are cultured in the absence of methotrexate. An analysis of the karyotypes of these resistant cells revealed the presence of numerous double minute chromosomes. We observed essentially identical kinetics of loss of dihydrofolate reductase gene sequences in total deoxyribonucleic acid and in deoxyribonucleic acid from fractions enriched in double minute chromosomes and in the numbers of double minute chromosomes per cell during reversion to methotrexate sensitivity, and this suggested that unstably amplified gene sequences were localized on double minute chromosomes. This conclusion ws also supported by an analysis of cell populations sorted according to dihydrofolate reductase enzyme contents, in which relative gene amplification and double minute chromosome content were related proportionally.
Deoxyribonucleic acid-mediated gene transfer in mammalian cells: molecular analysis of unstable transformants and their progression to stability.
Authors: Scangos G A , Huttner K M , Juricek D K , Ruddle F H
Published: September 2015

To elucidate mechanisms involved in deoxyribonucleic acid-mediated gene transfer, we transferred the herpes simplex virus thymidine kinase gene (TK) into mouse Ltk- cells. Independent TK+ clones (transformants) and derivatives of each were tested for phenotypic expression and the presence and arrangement of TK sequences. Initially, transformants expressed viral TK unstable, with 10% of the cells in each generation losing both the TK+ phenotype and virally derived TK sequences. After a prolonged period in culture, stable subpopulations arose from which the TK+ phenotype and viral sequences were no longer lost at detectable frequency. Analysis of unstable cell populations indicated that individual viral deoxyribonucleic acid molecules were reduced in size, but were linked to other deoxyribonucleic acid to form molecules large enough to be precipitated in a Hirt fractionation. We term these molecules transgenomes. Analysis of independent unstable subclones derived from the primary transformants demonstrated that individual transgenomes could contain multiple copies of the viral TK sequences. Recipient cell lines frequently possessed more than one type of transgenome and possibly multiple copies per cell of each type. Stable derivatives possessed only one of the transgenomes present in the unstable parent, and these sequences were associated with a recipient cell chromosome.
Chinese hamster ovary cells replicate adenovirus deoxyribonucleic acid.
Authors: Longiaru M , Horwitz M S
Published: September 2015

Chinese hamster ovary (CHO) cells infected with adenovirus type 2 (Ad2) produced amounts of viral deoxyribonucleic acid (DNA) equal to that synthesized in permissively infected HeLa cells. However, there was 6,000-fold less virion produced in CHO cells. Since the structural viral polypeptides were not detected by pulse-labeling CHO cells at various times postinfection, the block in virion formation is located between the synthesis of viral DNA and late proteins. Extracts of CHO cells could also function in a recently reported in vitro Ad2 DNA synthesis system which is dependent upon the addition of exogenous Ad2 DNA covalently linked to a 5'-terminal protein (Ikeda et al., Proc. Natl. Acad. Sci. U.S.A. 77:5827-5831, 1980). Extracts of infected CHO cytoplasm were able to complement uninfected CHO nuclear extracts to synthesize viral DNA on Ad2 templates. This in vitro replication system has the potential to probe host DNA synthesis requirements as well as viral factors.
Intracisternal A-particle genes in Mus musculus: a conserved family of retrovirus-like elements.
Authors: Kuff E L , Smith L A , Lueders K K
Published: September 2015

The structural organization of intracisternal A-particle genes has been studied, using isolates from a mouse gene library in lambda phage Charon 4A. The predominant gene form among the isolates was 7.3 kilobases (kb) in length. R-loops between the 7-kb (35S) A-particle genomic ribonucleic acid and several of these genes were colinear, with no visible evidence of intervening deoxyribonucleic acid sequences. One recombinant was found with an A-particle gene that contained a 1.7-kb deletion. Using the deletion as a reference, the deoxyribonucleic acid and ribonucleic acid homology regions were localized with respect to one another and to the restriction map: the 5' terminus of the ribonucleic acid was several hundred base pairs within the 5' end of the deoxyribonucleic acid homology region. Restriction endonuclease fragments encompassing the 5' and 3' regions of one 7.3-kb gene were separately subcloned into pBR322. Heteroduplexes between the two subclones revealed an approximately 300-base pair segment of terminally redundant sequences. The cloned 3' fragment hybridized with restriction fragments from the 5' end of several other A-particle genes, demonstrating the presence of common (though not necessarily identical) terminally repeated sequences. A-particle genes varied in the occurrence of specific restriction sites at characteristic internal loci. However, heteroduplexes between several variant 7.3-kb genes showed continuous homology regions even when spread under stringent hybridization conditions. The relative abundance of restriction site variants was highly conserved in 12 laboratory strains of Mus musculus, in embryonic and adult tissues of a single inbred strain, and in the SC-1 cell line of feral mouse origin, but appeared to differ in a feral Japanese substrain, Mus musculus molossinus. Some evidence suggests that subsets of A-particle genes may have similar flanking sequences. The results are discussed in terms of the evolution of this multigene family.
Rapid cell surface appearance of endocytic membrane proteins in Chinese hamster ovary cells.
Authors: Storrie B , Dreesen T D , Maurey K M
Published: September 2015

Lactoperoxidase was used to selectively radiolabel endocytic membrane. CHO cells were incubated with enzyme at 37 degrees C for 10 min to permit lactoperoxidase internalization. Radioiodination was done at 4 degrees C. About 90% of the radioiodinated products pelleted at 100,000 X g. From 12 to 15 different electrophoretic species were detected by one-dimensional gel electrophoresis. When cells labeled by internalized lactoperoxidase were warmed to 37 degrees C, the incorporated radioactivity was lost in a biphasic manner with an overall t1/2 of approximately 20 h. Upon warming cells to 37 degrees C, the labeled species became sensitive to pronase or trypsin digestion. The increase in protease sensitivity was progressive over a 10- to 20-min period. Maximally 45% of the initially intracellular radiolabel could be released. A digest of exterior-radioiodinated cells released 50% of the incorporated radioiodine. These observations strongly suggest a rapid shuttling of approximately 90% of the radioiodinated membrane species initially present within the cell to the cell surface.
In vitro translation of the intermediate filament proteins desmin and vimentin.
Authors: O'Connor C M , Asai D J , Flytzanis C N , Lazarides E
Published: September 2015

Polyadenylated ribonucleic acid (RNA) was isolated from chicken skeletal and smooth muscle and translated in a cell-free rabbit reticulocyte system. Both types of muscle tissue contain messenger RNAs that code for the intermediate filament proteins desmin and vimentin, and the relative concentrations of the two translation products reflect the prevalence of the two proteins in vivo. Desmin synthesis represents a greater proportion of the total protein synthesis from smooth muscle RNA than from skeletal muscle RNA, whereas the converse is true of vimentin synthesis. Fractionation of the RNA on formamide-containing sucrose gradients before translation indicates that the desmin messenger RNA is larger than the vimentin messenger RNA and contains an extensive noncoding segment. The desmin and vimentin messages code predominantly for the non-phosphorylated forms of desmin and vimentin. However, the ratio of phosphorylated to unphosphorylated forms of the proteins could be increased by adding cyclic adenosine monophosphate-dependent kinase activity to the translation mixtures. These results suggest that desmin and vimentin are each synthesized from a single messenger RNA species and that posttranslational phosphorylation generates the additional isoelectric variants of each which are observed in vivo.
The aromatic amino acid pathway branches at L-arogenate in Euglena gracilis.
Authors: Byng G S , Whitaker R J , Shapiro C L , Jensen R A
Published: September 2015

The recently characterized amino acid L-arogenate (Zamir et al., J. Am. Chem. Soc. 102:4499-4504, 1980) may be a precursor of either L-phenylalanine or L-tyrosine in nature. Euglena gracilis is the first example of an organism that uses L-arogenate as the sole precursor of both L-tyrosine and L-phenylalanine, thereby creating a pathway in which L-arogenate rather than prephenate becomes the metabolic branch point. E. gracilis ATCC 12796 was cultured in the light under myxotrophic conditions and harvested in late exponential phase before extract preparation for enzymological assays. Arogenate dehydrogenase was dependent upon nicotinamide adenine dinucleotide phosphate for activity. L-Tyrosine inhibited activity effectively with kinetics that were competitive with respect to L-arogenate and noncompetitive with respect to nicotinamide adenine dinucleotide phosphate. The possible inhibition of arogenate dehydratase by L-phenylalanine has not yet been determined. Beyond the latter uncertainty, the overall regulation of aromatic biosynthesis was studied through the characterization of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and chorismate mutase. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase was subject to noncompetitive inhibition by L-tyrosine with respect to either of the two substrates. Chorismate mutase was feedback inhibited with equal effectiveness by either L-tyrosine or L-phenylalanine. L-Tryptophan activated activity of chorismate mutase, a pH-dependent effect in which increased activation was dramatic above pH 7.8 L-Arogenate did not affect activity of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase or of chorismate mutase. Four species of prephenate aminotransferase activity were separated after ion-exchange chromatography. One aminotransferase exhibited a narrow range of substrate specificity, recognizing only the combination of L-glutamate with prephenate, phenylpyruvate, or 4-hydroxyphenylpyruvate. Possible natural relationships between Euglena spp. and fungi previously considered in the literature are discussed in terms of data currently available to define enzymological variation in the shikimate pathway.
SUC1 gene of Saccharomyces: a structural gene for the large (glycoprotein) and small (carbohydrate-free) forms of invertase.
Authors: Rodriguez L , Lampen J O , MacKay V L
Published: September 2015

Saccharomyces cerevisiae revertant strain D10-ER1 has been shown to contain thermosensitive forms of the large (glycoprotein) and small (carbohydrate-free) invertases and a very low level of the small enzyme, along with a wild-type level of the large form (T. Mizunaga et al., Mol. Cell. Biol. 1:460-468, 1981). These characteristics cosegregated in crosses of the revertant strain with wild-type sucrose-fermenting (SUC1) or nonfermenting (suc0) strains. In addition, there is tight linkage between sucrose and maltose fermentation in revertant D10-ER1 (characteristic of the SUC1 and MAL1 genes). From this we infer that a single reversion event is responsible for the several changes observed in D10-ER1, and that this mutation maps within or very close to the SUC1 gene present in the ancestor strain 4059-358D. The revertant SUC1 allele in D10-ER1 (termed SUC1-R1) was expressed independently of the wild-type SUC1 gene when both were present in diploid cells. Diploids carrying only the wild-type or the mutant genes synthesized invertases with the characteristics of the parental Suc+ haploids. The possibility that a modifier gene was responsible for the alterations in the invertases of revertant D10-ER1 was ruled out by appropriate crosses. We conclude that SUC1 is a structural gene that codes for both the large and the small forms of invertase and suggest that SUC2 through SUC5 are structural genes as well.
Isolation and characterization of dexamethasone-resistant mutants from human lymphoid cell line CEM-C7.
Authors: Harmon J M , Thompson E B
Published: September 2015

Fifty-four independent dexamethasone-resistant clones were isolated from the clonal, glucocorticoid-sensitive human leukemic T-cell line CEM-C7. Resistance to 1 microM dexamethasone was acquired spontaneously at a rate of 2.6 X 10(-5) per cell per generation as determined by fluctuation analysis. After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the phenotypic expression time for dexamethasone resistance was determined to be 3 days. Spontaneous acquisition of resistance to 0.1 mM 6-thioguanine appeared to occur at a much slower rate, 1.6 X 10(-6) per cell per generation. However, the expression time after MNNG mutagenesis for this resistant phenotype was greater than 11 days, suggesting that the different rates of acquisition for the two phenotypes measured by fluctuation analysis were the results of the disparate expression times. The mutagens ICR 191 and MNNG were effective in increasing the dexamethasone-resistant fraction of cells in mutagenized cultures; ICR 191 produced a 35.6-fold increase, and MNNG produced an 8.5-fold increase. All the spontaneous dexamethasone-resistant clones contained glucocorticoid receptors, usually less than half of the amount found in the parental clone. They are therefore strikingly different from dexamethasone-resistant clones derived from the mouse cell lines S49 and W7. Dexamethasone-resistant clones isolated after mutagenesis of CEM-C7 contained, on the average, lower concentrations of receptor than did those isolated spontaneously, and one clone contained no detectable receptor. These results are consistent with a mutational origin for dexamethasone resistance in these human cells at a haploid or functionally hemizygous locus. They also suggest that this is a useful system for mutation assay.
Enhancement of cytotoxicities of ricin and Pseudomonas toxin in Chinese hamster ovary cells by nigericin.
Authors: Ray B , Wu H C
Published: September 2015

Nigericin and monensin, ionophores for Na+ and K+, have been found to enhance the cytotoxicities of abrin, ricin, and Pseudomonas aeruginosa exotoxin A in Chinese hamster ovary (CHO) cells. They do not affect the cytotoxicity of diphtheria toxin in the same cell line. Maximal sensitization of the CHO cells toward ricin and Pseudomonas toxin requires preculture of CHO cells in the presence of nigericin. Inhibition of protein synthesis in CHO cells by ricin or Pseudomonas toxin is also enhanced by preculture of CHO cells in the presence of nigericin. These results suggest a common step in the intoxication process of ricin and Pseudomonas toxin, the rate of which is facilitated by pretreatment with nigericin. This step is, however, not shared by the intoxication of CHO cells with diphtheria toxin.
Enhanced internalization of ricin in nigericin-pretreated Chinese hamster ovary cells.
Authors: Ray B , Wu H C
Published: September 2015

Biochemical and electron microscopic autoradiographic studies with [125I] ricin have revealed that nigericin-pretreated Chinese hamster ovary cells are more efficient than untreated cells in the internalization of the toxin into the cells. These results suggest that the enhanced rate of internalization of ricin in nigericin-pretreated cells may account for the enhancement of cytotoxicity of ricin in Chinese hamster ovary cells by nigericin.