bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a novel phosphorylation site in the sodium–chloride cotransporter, NCC
Authors: Rosenbaek L. L. , Assentoft M. , Pedersen N. B. , MacAulay N. , Fenton R. A. 
Published: September 2012

Key points
The sodium–chloride cotransporter, NCC, is essential for renal electrolyte balance and its function can be regulated by protein phosphorylation
Here we report the role and regulation of a novel phosphorylation site in NCC at Ser124
Ser124 phosphorylation plays a role in mediating full NCC transport activity, but does not seem to be involved in NCC trafficking
Various physiological stimuli such as vasopressin and aldosterone regulate the abundance of the Ser124 phosphorylation status and other phosphorylation sites in NCC
Unlike other known phosphorylation sites in NCC, the STE20/SPS1‐related proline–alanine‐rich kinase and oxidative stress‐response kinases (SPAK and OSR1) were not able to phosphorylate NCC at Ser124
The results demonstrate that phosphorylation of NCC is a major factor in determining the function of NCC under various physiological conditions
Abstract  The sodium–chloride cotransporter, NCC, is essential for renal electrolyte balance. NCC function can be modulated by protein phosphorylation. In this study, we characterized the role and physiological regulation of a novel phosphorylation site in NCC at Ser124 (S124). Novel phospho‐specific antibodies targeting pS124‐NCC demonstrated a band of 160 kDa in the kidney cortex, but not medulla, which was preabsorbed by a corresponding phosphorylated peptide. Confocal microscopy with kidney tubule segment‐specific markers localized pS124‐NCC to all distal convoluted tubule cells. Double immunogold electron microscopy demonstrated that pS124‐NCC co‐localized with total NCC in the apical plasma membrane of distal convoluted tubule cells and intracellular vesicles. Acute treatment of Munich–Wistar rats or vasopressin‐deficient Brattleboro rats with the vasopressin type 2 receptor‐specific agonist dDAVP significantly increased pS124‐NCC abundance, with no changes in total NCC plasma membrane abundance. pS124‐NCC levels also increased in abundance in rats after stimulation of the renin–angiotensin–aldosterone system by dietary low sodium intake. In contrast to other NCC phosphorylation sites, the STE20/SPS1‐related proline–alanine‐rich kinase and oxidative stress‐response kinases (SPAK and OSR1) were not able to phosphorylate NCC at S124. Protein kinase arrays identified multiple kinases that were able to bind to the region surrounding S124. Four of these kinases (IRAK2, CDK6/Cyclin D1, NLK and mTOR/FRAP) showed weak but significant phosphorylation activity at S124. In oocytes, 36Cl uptake studies combined with biochemical analysis showed decreased activity of plasma membrane‐associated NCC when replacing S124 with alanine (A) or aspartic acid (D). In novel tetracycline‐inducible MDCKII‐NCC cell lines, S124A and S124D mutants were able to traffic to the plasma membrane similarly to wildtype NCC.
Novel Method To Assess Antiretroviral Target Trough Concentrations UsingIn VitroSusceptibility Data
Authors: Acosta Edward P. , Limoli Kay L. , Trinh Lan , Parkin Neil T. , King Jennifer R. , Weidler Jodi M. , Ofotokun Ighovwerha , Petropoulos Christos J. 
Published: September 2012

ABSTRACTDurable suppression of HIV-1 replication requires the establishment of antiretroviral drug concentrations that exceed the susceptibility of the virus strain(s) infecting the patient. Minimum plasma drug concentrations (Ctrough) are correlated with response, but determination of targetCtroughvalues is hindered by a paucity ofin vivoconcentration-response data. In the absence of these data,in vitrosusceptibility measurements, adjusted for serum protein binding, can provide estimations of suppressivein vivodrug concentrations. We derived serum protein binding correction factors (PBCF) for protease inhibitors, nonnucleoside reverse transcriptase inhibitors, and an integrase inhibitor by measuring the effect of a range of human serum concentrations onin vitrodrug susceptibility measured with the PhenoSense HIV assay. PBCFs corresponding to 100% HS were extrapolated using linear regression and ranged from 1.4 for nevirapine to 77 for nelfinavir. Using the mean 95% inhibitory concentration (IC95) for ≥1,200 drug-susceptible viruses, we calculated protein-bound IC95(PBIC95) values. PBIC95values were concordant with the minimum effectiveCtroughvalues that were established in well-designed pharmacodynamic studies (e.g., indinavir, saquinavir, and amprenavir). In other cases, the PBIC95values were notably lower (e.g., darunavir, efavirenz, and nevirapine) or higher (nelfinavir and etravirine) than existing target recommendations. The establishment of PBIC95values as described here provides a convenient and standardized approach for estimation of the minimum drug exposure that is required to maintain viral suppression and prevent the emergence of drug-resistant variants, particularly whenin vivoconcentration-response relationships are lacking.
Chitin Synthases with a Myosin Motor-Like Domain Control the Resistance of Aspergillus fumigatus to Echinocandins
Authors: Jiménez-Ortigosa Cristina , Aimanianda Vishukumar , Muszkieta Laetitia , Mouyna Isabelle , Alsteens David , Pire Stéphane , Beau Remi , Krappmann Sven , Beauvais Anne , Dufrêne Yves F. , Roncero César , Latgé Jean-Paul 
Published: September 2012

ABSTRACT

Aspergillus fumigatus
has two chitin synthases (
CSMA
and
CSMB
) with a myosin motor-like domain (MMD) arranged in a head-to-head configuration. To understand the function of these chitin synthases, single and double
csm
mutant strains were constructed and analyzed. Although there was a slight reduction in mycelial growth of the mutants, the total chitin synthase activity and the cell wall chitin content were similar in the mycelium of all of the mutants and the parental strain. In the conidia, chitin content in the Δ
csmA
strain cell wall was less than half the amount found in the parental strain. In contrast, the Δ
csmB
mutant strain and, unexpectedly, the Δ
csmA
/Δ
csmB
mutant strain did not show any modification of chitin content in their conidial cell walls. In contrast to the hydrophobic conidia of the parental strain, conidia of all of the
csm
mutants were hydrophilic due to the presence of an amorphous material covering the hydrophobic surface-rodlet layer. The deletion of
CSM
genes also resulted in an increased susceptibility of resting and germinating conidia to echinocandins. These results show that the deletion of the
CSMA
and
CSMB
genes induced a significant disorganization of the cell wall structure, even though they contribute only weakly to the overall cell wall chitin synthesis.
Cell Envelope Stress Response in Cell Wall-Deficient L-Forms of Bacillus subtilis
Authors: Wolf Diana , Domínguez-Cuevas Patricia , Daniel Richard A. , Mascher Thorsten 
Published: September 2012

ABSTRACT

L-forms are cell wall-deficient bacteria that can grow and proliferate in osmotically stabilizing media. Recently, a strain of the Gram-positive model bacterium
Bacillus subtilis
was constructed that allowed controlled switching between rod-shaped wild-type cells and corresponding L-forms. Both states can be stably maintained under suitable culture conditions. Because of the absence of a cell wall, L-forms are known to be insensitive to β-lactam antibiotics, but reports on the susceptibility of L-forms to other antibiotics that interfere with membrane-anchored steps of cell wall biosynthesis are sparse, conflicting, and strongly influenced by strain background and method of L-form generation. Here we investigated the response of
B. subtilis
to the presence of cell envelope antibiotics, with regard to both antibiotic resistance and the induction of the known LiaRS- and BceRS-dependent cell envelope stress biosensors. Our results show that
B. subtilis
L-forms are resistant to antibiotics that interfere with the bactoprenol cycle, such as bacitracin, vancomycin, and mersacidin, but are hypersensitive to nisin and daptomycin, which both affect membrane integrity. Moreover, we established a
lacZ
-based reporter gene assay for L-forms and provide evidence that LiaRS senses its inducers indirectly (damage sensing), while the Bce module detects its inducers directly (drug sensing).
Comparative Pharmacodynamics of the New Oxazolidinone Tedizolid Phosphate and Linezolid in a Neutropenic Murine Staphylococcus aureus Pneumonia Model
Authors: Lepak Alexander J. , Marchillo Karen , Pichereau Solen , Craig William A. , Andes David R. 
Published: September 2012

ABSTRACT

Tedizolid phosphate (TR-701) is a novel oxazolidinone prodrug (converted to the active form tedizolid [TR-700]) with potent
Staphylococcus aureus
activity. The current studies characterized and compared the
in vivo
pharmacokinetic/pharmacodynamic (PD) characteristics of TR-701/TR-700 and linezolid against methicillin-susceptible
S. aureus
(MSSA) and methicillin-resistant
S. aureus
(MRSA) in the neutropenic murine pneumonia model. The pharmacokinetic properties of both drugs were linear over a dose range of 0.625 to 40 mg/kg of body weight. Protein binding was 30% for linezolid and 85% for TR-700. Mice were infected with one of 11 isolates of
S. aureus
, including MSSA and community- and hospital-acquired MRSA strains. Each drug was administered by oral-gastric gavage every 12 h (q12h). The dosing regimens ranged from 1.25 to 80 mg/kg/12 h for linezolid and 0.625 to 160 mg/kg/12 h for TR-701. At the start of therapy, mice had 6.24 ± 0.40 log
10
CFU/lungs, which increased to 7.92 ± 1.02 log
10
CFU/lungs in untreated animals over a 24-h period. A sigmoid maximum-effect (
E
max
) model was used to determine the antimicrobial exposure associated with net stasis (static dose [SD]) and 1-log-unit reduction in organism relative to the burden at the start of therapy. The static dose pharmacodynamic targets for linezolid and TR-700 were nearly identical, at a free drug (non-protein-bound) area under the concentration-time curve over 24 h in the steady state divided by the MIC (AUC/MIC ratio) of 19 and 20, respectively. The 1-log-unit kill endpoints were also similar, at 46.1 for linezolid and 34.6 for TR-700. The exposure targets were also comparable for both MSSA and MRSA isolates. These dosing goals support further clinical trial examination of TR-701 in MSSA and MRSA pneumonia.
Inhibition of Staphylococcus epidermidis Biofilm by Trimethylsilane Plasma Coating
Authors: Ma Yibao , Chen Meng , Jones John E. , Ritts Andrew C. , Yu Qingsong , Sun Hongmin 
Published: September 2012

ABSTRACT

Biofilm formation on implantable medical devices is a major impediment to the treatment of nosocomial infections and promotes local progressive tissue destruction.
Staphylococcus epidermidis
infections are the leading cause of biofilm formation on indwelling devices. Bacteria in biofilms are highly resistant to antibiotic treatment, which in combination with the increasing prevalence of antibiotic resistance among human pathogens further complicates treatment of biofilm-related device infections. We have developed a novel plasma coating technology. Trimethylsilane (TMS) was used as a monomer to coat the surfaces of 316L stainless steel and grade 5 titanium alloy, which are widely used in implantable medical devices. The results of biofilm assays demonstrated that this TMS coating markedly decreased
S. epidermidis
biofilm formation by inhibiting the attachment of bacterial cells to the TMS-coated surfaces during the early phase of biofilm development. We also discovered that bacterial cells on the TMS-coated surfaces were more susceptible to antibiotic treatment than their counterparts in biofilms on uncoated surfaces. These findings suggested that TMS coating could result in a surface that is resistant to biofilm development and also in a bacterial community that is more sensitive to antibiotic therapy than typical biofilms.
Serine-Type Carboxypeptidase KexA of Aspergillus oryzae Has Broader Substrate Specificity than Saccharomyces cerevisiae Kex1 and Is Required for Normal Hyphal Growth and Conidiation
Authors: Morita Hiroto , Tomita Sayako , Maeda Hiroshi , Okamoto Ayako , Yamagata Youhei , Kusumoto Ken-Ichi , Amano Hitoshi , Ishida Hiroki , Takeuchi Michio 
Published: September 2012

ABSTRACT

Aspergillus oryzae
has an ortholog of
Saccharomyces cerevisiae KEX1
, termed
kexA
. A truncated form of KexA protein showed serine-type carboxypeptidase activity and somewhat broader substrate specificity than Kex1 protease. Furthermore, our results indicated that KexA is required for normal growth of
A. oryzae
and that it might be involved in hyphal branching.
Increased Resistance to Multiple Antimicrobials and Altered Resistance Gene Expression in CMY-2-Positive Salmonella enterica following a Simulated Patient Treatment with Ceftriaxone
Authors: Hamilton Russell D. , Hulsebus Holly J. , Akbar Samina , Gray Jeffrey T. 
Published: September 2012

ABSTRACTSalmonellosis is one of the most common causes of food-borne disease in the United States. Increasing antimicrobial resistance and corresponding increases in virulence present serious challenges. Currently, empirical therapy for invasiveSalmonella entericainfection includes either ceftriaxone or ciprofloxacin (E. L. Hohmann, Clin. Infect. Dis. 32:263–269, 2001). TheblaCMY-2gene confers resistance to ceftriaxone, the antimicrobial of choice for pediatric patients with invasiveSalmonella entericainfections, making these infections especially dangerous (J. M. Whichard et al., Emerg. Infect. Dis. 11:1464–1466, 2005). We hypothesized thatblaCMY-2-positiveSalmonella entericawould exhibit increased MICs to multiple antimicrobial agents and increased resistance gene expression following exposure to ceftriaxone using a protocol that simulated a patient treatmentin vitro. SevenSalmonella entericastrains survived a simulated patient treatmentin vitroand, following treatment, exhibited a significantly increased ceftriaxone MIC. Not only would these isolates be less responsive to further ceftriaxone treatment, but because theblaCMY-2genes are commonly located on large, multidrug-resistant plasmids, increased expression of theblaCMY-2gene may be associated with increased expression of other drug resistance genes located on the plasmid (N. D. Hanson and C. C. Sanders, Curr. Pharm. Des. 5:881–894, 1999). The results of this study demonstrate that a simulated patient treatment with ceftriaxone can alter the expression of antimicrobial resistance genes, includingblaCMY-2andfloRinS. entericaserovar Typhimurium andS. entericaserovar Newport. Additionally, we have shown increased MICs following a simulated patient treatment with ceftriaxone for tetracycline, amikacin, ceftriaxone, and cefepime, all of which have resistance genes commonly located on CMY-2 plasmids. The increases in resistance observed are significant and may have a negative impact on both public health and antimicrobial resistance ofSalmonella enterica.
Impact of Vitamin B
12
on Formation of the Tetrachloroethene Reductive Dehalogenase in Desulfitobacterium hafniense Strain Y51
Authors: Reinhold Anika , Westermann Martin , Seifert Jana , von Bergen Martin , Schubert Torsten , Diekert Gabriele 
Published: September 2012

ABSTRACT

Corrinoids are essential cofactors of reductive dehalogenases in anaerobic bacteria. Microorganisms mediating reductive dechlorination as part of their energy metabolism are either capable of
de novo
corrinoid biosynthesis (e.g.,
Desulfitobacterium
spp.) or dependent on exogenous vitamin B
12
(e.g.,
Dehalococcoides
spp.). In this study, the impact of exogenous vitamin B
12
(cyanocobalamin) and of tetrachloroethene (PCE) on the synthesis and the subcellular localization of the reductive PCE dehalogenase was investigated in the Gram-positive
Desulfitobacterium hafniense
strain Y51, a bacterium able to synthesize corrinoids
de novo
. PCE-depleted cells grown for several subcultivation steps on fumarate as an alternative electron acceptor lost the tetrachloroethene-reductive dehalogenase (PceA) activity by the transposition of the
pce
gene cluster. In the absence of vitamin B
12
, a gradual decrease of the PceA activity and protein amount was observed; after 5 subcultivation steps with 10% inoculum, more than 90% of the enzyme activity and of the PceA protein was lost. In the presence of vitamin B
12
, a significant delay in the decrease of the PceA activity with an ∼90% loss after 20 subcultivation steps was observed. This corresponded to the decrease in the
pceA
gene level, indicating that exogenous vitamin B
12
hampered the transposition of the
pce
gene cluster. In the absence or presence of exogenous vitamin B
12
, the intracellular corrinoid level decreased in fumarate-grown cells and the PceA precursor formed catalytically inactive, corrinoid-free multiprotein aggregates. The data indicate that exogenous vitamin B
12
is not incorporated into the PceA precursor, even though it affects the transposition of the
pce
gene cluster.
Enhancement of Acetic Acid Tolerance in Saccharomyces cerevisiae by Overexpression of the
HAA1
Gene, Encoding a Transcriptional Activator
Authors: Tanaka Koichi , Ishii Yukari , Ogawa Jun , Shima Jun 
Published: September 2012

ABSTRACT

Haa1 is a transcriptional activator required for
Saccharomyces cerevisiae
adaptation to weak acids. Here we show that the constitutive
HAA1
-overexpressing strain acquired a higher level of acetic acid tolerance. Under conditions of acetic acid stress, the intracellular level of acetic acid was significantly lower in
HAA1
-overexpressing cells than in the wild-type cells.
Overexpression of a Novel Biotrophy-Specific Colletotrichum truncatum Effector, CtNUDIX, in Hemibiotrophic Fungal Phytopathogens Causes Incompatibility with Their Host Plants
Authors: Bhadauria Vijai , Banniza Sabine , Vandenberg Albert , Selvaraj Gopalan , Wei Yangdou 
Published: September 2012

ABSTRACTThe hemibiotrophic fungusColletotrichum truncatumcauses anthracnose disease on lentils and a few other grain legumes. It shows initial symptomless intracellular growth, where colonized host cells remain viable (biotrophy), and then switches to necrotrophic growth, killing the colonized host plant tissues. Here, we report a novel effector gene,CtNUDIX, fromC. truncatumthat is exclusively expressed during the late biotrophic phase (before the switch to necrotrophy) and elicits a hypersensitive response (HR)-like cell death in tobacco leaves transiently expressing the effector. CtNUDIX homologs, which contain a signal peptide and a Nudix hydrolase domain, may be unique to hemibiotrophic fungal and fungus-like plant pathogens. CtNUDIX lacking a signal peptide or a Nudix motif failed to induce cell death in tobacco. Expression of CtNUDIX:eGFP in tobacco suggested that the fusion protein might act on the host cell plasma membrane. Overexpression ofCtNUDIXinC. truncatumand the rice blast pathogen,Magnaporthe oryzae, resulted in incompatibility with the hosts lentil and barley, respectively, by causing an HR-like response in infected host cells associated with the biotrophic invasive hyphae. These results suggest thatC. truncatumand possiblyM. oryzaeelicit cell death to signal the transition from biotrophy to necrotrophy.
Campylobacter jejuni Outer Membrane Vesicles Play an Important Role in Bacterial Interactions with Human Intestinal Epithelial Cells
Authors: Elmi Abdi , Watson Eleanor , Sandu Pamela , Gundogdu Ozan , Mills Dominic C. , Inglis Neil F. , Manson Erin , Imrie Lisa , Bajaj-Elliott Mona , Wren Brendan W. , Smith David G. E. , Dorrell Nick 
Published: September 2012

ABSTRACT

Campylobacter jejuni
is the most prevalent cause of food-borne gastroenteritis in the developed world; however, the molecular basis of pathogenesis is unclear. Secretion of virulence factors is a key mechanism by which enteric bacterial pathogens interact with host cells to enhance survival and/or damage the host. However,
C. jejuni
lacks the virulence-associated secretion systems possessed by other enteric pathogens. Many bacterial pathogens utilize outer membrane vesicles (OMVs) for delivery of virulence factors into host cells. In the absence of prototypical virulence-associated secretion systems, OMVs could be an important alternative for the coordinated delivery of
C. jejuni
proteins into host cells. Proteomic analysis of
C. jejuni
11168H OMVs identified 151 proteins, including periplasmic and outer membrane-associated proteins, but also many determinants known to be important in survival and pathogenesis, including the cytolethal distending toxin (CDT).
C. jejuni
OMVs contained 16
N
-linked glycoproteins, indicating a delivery mechanism by which these periplasm-located yet immunogenic glycoproteins can interact with host cells.
C. jejuni
OMVs possess cytotoxic activity and induce a host immune response from T84 intestinal epithelial cells (IECs), which was not reduced by OMV pretreatment with proteinase K or polymyxin B prior to coincubation with IECs. Pretreatment of IECs with methyl-beta-cyclodextrin partially blocks OMV-induced host immune responses, indicating a role for lipid rafts in host cell plasma membranes during interactions with
C. jejuni
OMVs. OMVs isolated from a
C. jejuni
11168H
cdtA
mutant induced interleukin-8 (IL-8) to the same extent as did wild-type OMVs, suggesting OMV induction of IL-8 is independent of CDT.
Global Effect of Interleukin-10 on the Transcriptional Profile Induced by Neisseria meningitidis in Human Monocytes
Authors: Gopinathan Unni , Øvstebø Reidun , Olstad Ole Kristoffer , Brusletto Berit , Dalsbotten Aass Hans Christian , Kierulf Peter , Brandtzaeg Petter , Berg Jens Petter 
Published: September 2012

ABSTRACTIn meningococcal septic shock, the dominant inducer of inflammation is lipopolysaccharide (LPS) in the outer membrane ofNeisseria meningitidis, while interleukin-10 (IL-10) is the principal anti-inflammatory cytokine. We have used microarrays and Ingenuity Pathway Analysis to study the global effects of IL-10 on gene expression induced byN. meningitidis, after exposure of human monocytes (n= 5) for 3 h toN. meningitidis(106cells/ml), recombinant human IL-10 (rhIL-10) (25 ng/ml), andN. meningitidiscombined with rhIL-10.N. meningitidisand IL-10 differentially expressed 3,579 and 648 genes, respectively. IL-10 downregulated 125 genes which were upregulated byN. meningitidis, including NLRP3, the key molecule of the NLRP3 inflammasome. IL-10 also upregulated 270 genes which were downregulated byN. meningitidis, including members of the leukocyte immunuglobulin-like receptor (LIR) family. Fifty-three genes revealed a synergistically increased expression whenN. meningitidisand IL-10 were combined. AIM2 (the principal molecule of the AIM2 inflammasome) was among these genes (fold change [FC], 18.3 versus 7.4 and 9.4 after stimulation byN. meningitidisand IL-10, respectively). We detected reduced concentrations (92% to 40%) of six cytokines (IL-1b, IL-6, IL-8, tumor necrosis factor alpha [TNF-α], macrophage inflammatory protein alpha [MIP-α], MIP-β) in the presence of IL-10, compared with concentrations with stimulation byN. meningitidisalone. Our data analysis of the effects of IL-10 on gene expression induced byN. meningitidissuggests that high plasma levels of IL-10 in meningococcal septic shock plasma may have a profound effect on a variety of functions and cellular processes in human monocytes, including cell-to-cell signaling, cellular movement, cellular development, antigen presentation, and cell death.