FtsZ filament structures in different nucleotide states reveal the mechanism of assembly dynamics

Treadmilling protein filaments perform essential cellular functions by growing from one end while shrinking from the other, driven by nucleotide hydrolysis. Bacterial cell division relies on the primitive tubulin homolog FtsZ, a target for antibiotic discovery that assembles into single treadmilling filaments that hydrolyse GTP at an active site formed upon subunit association. We determined high-resolution filament structures of FtsZ from the pathogen Staphylococcus aureus in complex with different nucleotide analogs and cations, including mimetics of the ground and transition states of catalysis. Together with mutational and biochemical analyses, our structures reveal interactions made by the GTP γ-phosphate and Mg²⁺ at the subunit interface, a K⁺ ion stabilizing loop T7 for co-catalysis, new roles of key residues at the active site and a nearby crosstalk area, and rearrangements of a dynamic water shell bridging adjacent subunits upon GTP hydrolysis. We propose a mechanistic model that integrates nucleotide hydrolysis signaling with assembly-associated conformational changes and filament treadmilling. Equivalent assembly mechanisms may apply to more complex tubulin and actin cytomotive filaments that share analogous features with FtsZ.

Bacterial cell division critically relies on the tubulin homolog FtsZ, which assembles into filaments that treadmill, fuelled by GTP hydrolysis. This structural and biochemical study of FtsZ from Staphylocuccus aureus reveals the mechanism of GTP hydrolysis and its connection with filament dynamics.     

Expression of functional proteins of cDNA encoding rice nucleoside diphosphate kinase (NDK) in Escherichia coli and organ-related alteration of NDK activities during rice seed germination (Oryza sativa L.)

The GST (glutathione S-transferase)-NDK (nucleoside diphosphate kinase) fusion protein was expressed in Escherichia coli. The GST-NDK protein was capable of transferring -phosphate from ATP to nucleoside diphosphates such as GDP, CDP, TDP and UDP. Western blot analysis using anti-NDK antibody indicated that NDK in endosperm gradually decreased during 36 h of imbibition. On the contrary, NDK in embryo increased during the same period. NDK activities in both tissues were in accord with these observations. Whereas the NDK protein in roots of rice seedlings during 7 days of imbibition remained constant, in shoots it declined after 5 days of imbibition. Thus, NDK may play a significant role in the cellular event modulated by adenylate energy charge level.     

The Cell Division Protein FtsZ from Streptococcus pneumoniae Exhibits a GTPase Activity Delay

The cell division protein FtsZ assembles in vitro by a mechanism of cooperative association dependent on GTP, monovalent cations, and Mg²⁺. We have analyzed the GTPase activity and assembly dynamics of Streptococcus pneumoniae FtsZ (SpnFtsZ). SpnFtsZ assembled in an apparently cooperative process, with a higher critical concentration than values reported for other FtsZ proteins. It sedimented in the presence of GTP as a high molecular mass polymer with a well defined size and tended to form double-stranded filaments in electron microscope preparations. GTPase activity depended on K⁺ and Mg²⁺ and was inhibited by Na⁺. GTP hydrolysis exhibited a delay that included a lag phase followed by a GTP hydrolysis activation step, until reaction reached the GTPase rate. The lag phase was not found in polymer assembly, suggesting a transition from an initial non-GTP-hydrolyzing polymer that switches to a GTP-hydrolyzing polymer, supporting models that explain FtsZ polymer cooperativity.     

FtsZ fiber bundling is triggered by a conformational change in bound GTP

Polymer formation by the essential FtsZ protein plays a crucial role in the cytokinesis of most prokaryotes. Lateral associations between these FtsZ polymers to form bundles or sheets are widely predicted to be extremely important for FtsZ function in vivo. We have carried out a study in vitro of FtsZ polymer formation and bundling using linear dichroism (LD) to assess structural properties of the polymers. We demonstrate proof-of-principle experiments to show that LD can be used as a technique to follow FtsZ polymerization, and we present the LD spectra of FtsZ polymers. Our subsequent examination of FtsZ polymer bundling induced by calcium reveals a substantial increase in the LD signal indicative of increased polymer length and rigidity. We also detect a specific conformational change in the guanine moiety associated with bundling, whereas the conformation and configuration of the FtsZ monomers within the polymer remain largely unchanged. We demonstrate that other divalent cations can induce this conformational change in FtsZ-bound GTP coincident with polymer bundling. Therefore, we present "flipping" of the guanine moiety in FtsZ-bound GTP as a mechanism that explains the link between reduced GTPase activity, increased polymer stability, and polymer bundling.     

Characterization of ftsZ Mutations that Render Bacillus subtilis Resistant to MinC

Background

Cell division in Bacillus subtilis occurs precisely at midcell. Positional control of cell division is exerted by two mechanisms: nucleoid occlusion, through Noc, which prevents division through nucleoids, and the Min system, where the combined action of the MinC, D and J proteins prevents formation of the FtsZ ring at cell poles or recently completed division sites.

Methodology/Principal Findings

We used a genetic screen to identify mutations in ftsZ that confer resistance to the lethal overexpression of the MinC/MinD division inhibitor. The FtsZ mutants were purified and found to polymerize to a similar or lesser extent as wild type FtsZ, and all mutants displayed reduced GTP hydrolysis activity indicative of a reduced polymerization turnover. We found that even though the mutations conferred in vivo resistance to MinC/D, the purified FtsZ mutants did not display strong resistance to MinC in vitro.

Conclusions/Significance

Our results show that in B. subtilis, overproduction of MinC can be countered by mutations that alter FtsZ polymerization dynamics. Even though it would be very likely that the FtsZ mutants found depend on other Z-ring stabilizing proteins such as ZapA, FtsA or SepF, we found this not to be the case. This indicates that the cell division process in B. subtilis is extremely robust.     

In Vitro Analysis of Fibronectin-Modified Titanium Surfaces

Background

Glow discharge plasma (GDP) procedure is an effective method for grafting various proteins, including albumin, type I collagen, and fibronectin, onto a titanium surface. However, the behavior and impact of titanium (Ti) surface modification is yet to be unraveled.

Purpose

The purpose of this study is to evaluate and analyze the biological properties of fibronectin-grafted Ti surfaces treated by GDP.

Materials and Methods

Grade II Ti discs were initially cleaned and autoclaved to obtain original specimens. Subsequently, the specimens were GDP treated and grafted with fibronectin to form Ar-GDP (Argon GDP treatment only) and GDP-fib (fibronectin coating following GDP treatment) groups. Blood coagulation test and MG-63 cell culture were performed to evaluate the biological effects on the specimen.

Results

There was no significant difference between Ar-GDP and GDP-fib groups in blood compatibility analysis. While in the MTT test, cellular proliferation was benefited from the presence of fibronectin coating. The numbers of cells on Ar-GDP and GDP-fib specimens were greater than those in the original specimens after 24 h of culturing.

Conclusions

GDP treatment combined with fibronectin grafting favored MG-63 cell adhesion, migration, and proliferation on titanium surfaces, which could be attributed to the improved surface properties.     

Non-hydrolysable GTP-gamma-S stabilizes the FtsZ polymer in a GDP-bound state

FtsZ, a tubulin homologue, forms a cytokinetic ring at the site of cell division in prokaryotes. The ring is thought to consist of polymers that assemble in a strictly GTP-dependent way. GTP, but not guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), has been shown to induce polymerization of FtsZ, whereas in vitro Ca2+ is known to inhibit the GTP hydrolysis activity of FtsZ. We have studied FtsZ dynamics at limiting GTP concentrations in the presence of 10 mM Ca2+. GTP and its non-hydrolysable analogue GTP-gamma-S bind FtsZ with similar affinity, whereas the non-hydrolysable analogue guanylyl-imidodiphosphate (GMP-PNP) is a poor substrate. Preformed FtsZ polymers can be stabilized by GTP-gamma-S and are destabilized by GDP. As more than 95% of the nucleotide associated with the FtsZ polymer is in the GDP form, it is concluded that GTP hydrolysis by itself does not trigger FtsZ polymer disassembly. Strikingly, GTP-gamma-S exchanges only a small portion of the FtsZ polymer-bound GDP. These data suggest that FtsZ polymers are stabilized by a small fraction of GTP-containing FtsZ subunits. These subunits may be located either throughout the polymer or at the polymer ends, forming a GTP cap similar to tubulin.     

The Nucleoid Occlusion SlmA Protein Accelerates the Disassembly of the FtsZ Protein Polymers without Affecting Their GTPase Activity

Division site selection is achieved in bacteria by different mechanisms, one of them being nucleoid occlusion, which prevents Z-ring assembly nearby the chromosome. Nucleoid occlusion in E. coli is mediated by SlmA, a sequence specific DNA binding protein that antagonizes FtsZ assembly. Here we show that, when bound to its specific target DNA sequences (SBS), SlmA reduces the lifetime of the FtsZ protofilaments in solution and of the FtsZ bundles when located inside permeable giant vesicles. This effect appears to be essentially uncoupled from the GTPase activity of the FtsZ protofilaments, which is insensitive to the presence of SlmA·SBS. The interaction of SlmA·SBS with either FtsZ protofilaments containing GTP or FtsZ oligomers containing GDP results in the disassembly of FtsZ polymers. We propose that SlmA·SBS complexes control the polymerization state of FtsZ by accelerating the disassembly of the FtsZ polymers leading to their fragmentation into shorter species that are still able to hydrolyze GTP at the same rate. SlmA defines therefore a new class of inhibitors of the FtsZ ring different from the SOS response regulator SulA and from the moonlighting enzyme OpgH, inhibitors of the GTPase activity. SlmA also shows differences compared with MinC, the inhibitor of the division site selection Min system, which shortens FtsZ protofilaments by interacting with the GDP form of FtsZ.     

Modification of β-Defensin-2 by Dicarbonyls Methylglyoxal and Glyoxal Inhibits Antibacterial and Chemotactic Function In Vitro

Background

Beta-defensins (hBDs) provide antimicrobial and chemotactic defense against bacterial, viral and fungal infections. Human β-defensin-2 (hBD-2) acts against gram-negative bacteria and chemoattracts immature dendritic cells, thus regulating innate and adaptive immunity. Immunosuppression due to hyperglycemia underlies chronic infection in Type 2 diabetes. Hyperglycemia also elevates production of dicarbonyls methylgloxal (MGO) and glyoxal (GO).

Methods

The effect of dicarbonyl on defensin peptide structure was tested by exposing recombinant hBD-2 (rhBD-2) to MGO or GO with subsequent analysis by MALDI-TOF MS and LC/MS/MS. Antimicrobial function of untreated rhBD-2 vs. rhBD-2 exposed to dicarbonyl against strains of both gram-negative and gram-positive bacteria in culture was determined by radial diffusion assay. The effect of dicarbonyl on rhBD-2 chemotactic function was determined by chemotaxis assay in CEM-SS cells.

Results

MGO or GO in vitro irreversibly adducts to the rhBD-2 peptide, and significantly reduces antimicrobial and chemotactic functions. Adducts derive from two arginine residues, Arg²² and Arg²³ near the C-terminus, and the N-terminal glycine (Gly¹). We show by radial diffusion testing on gram-negative E. coli and P. aeruginosa, and gram-positive S. aureus, and a chemotaxis assay for CEM-SS cells, that antimicrobial activity and chemotactic function of rhBD-2 are significantly reduced by MGO.

Conclusions

Dicarbonyl modification of cationic antimicrobial peptides represents a potential link between hyperglycemia and the clinical manifestation of increased susceptibility to infection, protracted wound healing, and chronic inflammation in undiagnosed and uncontrolled Type 2 diabetes.     

Single tryptophan mutants of FtsZ: Nucleotide binding/exchange and conformational transitions

Cell division protein FtsZ cooperatively self-assembles into straight filaments when bound to GTP. A set of conformational changes that are linked to FtsZ GTPase activity are involved in the transition from straight to curved filaments that eventually disassemble. In this work, we characterized the fluorescence of single Trp mutants as a reporter of the predicted conformational changes between the GDP- and GTP-states of Escherichia coli FtsZ. Steady-state fluorescence characterization showed the Trp senses different environments and displays low solvent accessibility. Time-resolved fluorescence data indicated that the main conformational changes in FtsZ occur at the interaction surface between the N and C domains, but also minor rearrangements were detected in the bulk of the N domain. Surprisingly, despite its location near the bottom protofilament interface at the C domain, the Trp 275 fluorescence lifetime did not report changes between the GDP and GTP states. The equilibrium unfolding of FtsZ features an intermediate that is stabilized by the nucleotide bound in the N-domain as well as by quaternary protein–protein interactions. In this context, we characterized the unfolding of the Trp mutants using time-resolved fluorescence and phasor plot analysis. A novel picture of the structural transition from the native state in the absence of denaturant, to the solvent-exposed unfolded state is presented. Taken together our results show that conformational changes between the GDP and GTP states of FtsZ, such as those observed in FtsZ unfolding, are restricted to the interaction surface between the N and C domains.     

FtsZ and bacterial cell division

In this review we describe proteins and supermolecular structures which take part in the division of bacterial cells. FtsZ, a eukaryotic tubulin homolog is a key cell division protein in most prokaryotes. FtsZ, as well as tubulin, is capable of binding and hydrolyzing GTP. The division of a bacterial cell begins with the forming of a so-called divisome. The basis of such a divisome is a contractile ring (Z ring) which encircles the cell about midcell. The Z-ring consists of a bundle of laterally bound protofilaments formed in result of FtsZ polymerization. Z-ring is rigidly bounded to the cytosolic side of the inner membrane with the participation of FtsA, ZipA, FtsW and many other divisome cell division proteins. The ring directs the process of cytokinesis transmitting constriction power to the membrane. The primary structures of the prokaryotic FtsZ family members significantly differ from eukaryotic tubulins except for the sites of GTP binding. There is a high degree of structural homology between these proteins in the region. FtsZ is one of the most conserved proteins in prokaryotes. However, ftsZ genes have not been found in several species of microorganisms with completely sequenced genomes. They include two species of mycoplasmas (Ureaplasma parvum and Mycoplasma mobile), Prostecobacter dejongeii, 10 species of chlamydia and 5 species of archaea. Consequently, these organisms divide without FtsZ participation. The genomes of U. parvum and M. mobile have many open reading frames which encode proteins with unknown functions. A comparison of the primary structures of these hypothetical proteins did not identify any known cell division proteins. We hypothesize that the process of cell division in these organisms should involve proteins similar to FtsZ in function and homologous to FtsZ or other cell division proteins in structure.     

FtsZ Polymerization Assays: Simple Protocols and Considerations

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long filaments in the presence of GTP in vitro, and polymerization is regulated by several accessory proteins. FtsZ polymerization has been extensively studied in vitro using basic methods including light scattering, sedimentation, GTP hydrolysis assays and electron microscopy. Buffer conditions influence both the polymerization properties of FtsZ, and the ability of FtsZ to interact with regulatory proteins. Here, we describe protocols for FtsZ polymerization studies and validate conditions and controls using Escherichia coli and Bacillus subtilis FtsZ as model proteins. A low speed sedimentation assay is introduced that allows the study of the interaction of FtsZ with proteins that bundle or tubulate FtsZ polymers. An improved GTPase assay protocol is described that allows testing of GTP hydrolysis over time using various conditions in a 96-well plate setup, with standardized incubation times that abolish variation in color development in the phosphate detection reaction. The preparation of samples for light scattering studies and electron microscopy is described. Several buffers are used to establish suitable buffer pH and salt concentration for FtsZ polymerization studies. A high concentration of KCl is the best for most of the experiments. Our methods provide a starting point for the in vitro characterization of FtsZ, not only from E. coli and B. subtilis but from any other bacterium. As such, the methods can be used for studies of the interaction of FtsZ with regulatory proteins or the testing of antibacterial drugs which may affect FtsZ polymerization.     

Adenine Nucleotide-dependent Regulation of Assembly of Bacterial Tubulin-like FtsZ by a Hypermorph of Bacterial Actin-like FtsA

Cytokinesis in bacteria depends upon the contractile Z ring, which is composed of dynamic polymers of the tubulin homolog FtsZ as well as other membrane-associated proteins such as FtsA, a homolog of actin that is required for membrane attachment of the Z ring and its subsequent constriction. Here we show that a previously characterized hypermorphic mutant FtsA (FtsA*) partially disassembled FtsZ polymers in vitro. This effect was strictly dependent on ATP or ADP binding to FtsA* and occurred at substoichiometric levels relative to FtsZ, similar to cellular levels. Nucleotide-bound FtsA* did not affect FtsZ GTPase activity or the critical concentration for FtsZ assembly but was able to disassemble preformed FtsZ polymers, suggesting that FtsA* acts on FtsZ polymers. Microscopic examination of the inhibited FtsZ polymers revealed a transition from long, straight polymers and polymer bundles to mainly short, curved protofilaments. These results indicate that a bacterial actin, when activated by adenine nucleotides, can modify the length distribution of bacterial tubulin polymers, analogous to the effects of actin-depolymerizing factor/cofilin on F-actin.Bacterial cell division requires a large number of proteins that colocalize to form a putative protein machine at the cell membrane (1). This machine, sometimes called the divisome, recruits enzymes to synthesize the septum cell wall and to initiate and coordinate the invagination of the cytoplasmic membrane (and in Gram-negative bacteria, the outer membrane). The most widely conserved and key protein for this process is FtsZ, a homolog of tubulin that forms a ring structure called the Z ring, which marks the site of septum formation (2, 3). Like tubulin, FtsZ assembles into filaments with GTP but does not form microtubules (4). The precise assembly state and conformation of these FtsZ filaments at the division ring is not clear, although recent electron tomography work suggests that the FtsZ ring consists of multiple short filaments tethered to the membrane at discrete junctures (5), which may represent points along the filaments bridged by membrane anchor proteins.In Escherichia coli, two of these anchor proteins are known. One of these, ZipA, is not well conserved but is an essential protein in E. coli. ZipA binds to the C-terminal tail of FtsZ (6–8), and purified ZipA promotes bundling of FtsZ filaments in vitro (9, 10). The other, FtsA, is also essential in E. coli and is more widely conserved among bacterial species. FtsA is a member of the HSP70/actin superfamily (11, 12), and like ZipA, it interacts with the C-terminal tail of FtsZ (7, 13–15). FtsA can self-associate (16, 17) and bind ATP (12, 18), but reports of ATPase activity vary, with Bacillus subtilis FtsA having high activity (19) and Streptococcus pneumoniae FtsA exhibiting no detectable activity (20). There are no reports of any other in vitro activities of FtsA, including effects on FtsZ assembly.Understanding how FtsA affects FtsZ assembly is important because FtsA has a number of key activities in the cell. It is required for recruitment of a number of divisome proteins (21, 22) and helps to tether the Z ring to the membrane via a C-terminal membrane-targeting sequence (23). FtsA, like ZipA and other divisome proteins, is necessary to activate the contraction of the Z ring (24, 25). In E. coli, the FtsA:FtsZ ratio is crucial for proper cell division, with either too high or too low a ratio inhibiting septum formation (26, 27). This ratio is roughly 1:5, with ∼700 molecules of FtsA and 3200 molecules of FtsZ per cell (28), which works out to concentrations of 1–2 and 5–10 μm, respectively.Another interesting property of FtsA is that single residue alterations in the protein can result in significant enhancement of divisome activity. For example, the R286W mutation of FtsA, also called FtsA*, can substitute for the native FtsA and divide the cell. However, this mutant FtsA causes E. coli cells to divide at less than 80% of their normal length (29) and allows efficient division of E. coli cells in the absence of ZipA (30), indicating that it has gain-of-function activity. FtsA* and other hypermorphic mutations such as E124A and I143L can also increase division activity in cells lacking other essential divisome components (31–33). The R286W and E124A mutants of FtsA also bypass the FtsA:FtsZ ratio rule, allowing cell division to occur at higher ratios than with WT² FtsA. This may be because the altered FtsA proteins self-associate more readily than WT FtsA, which may cause different changes in FtsZ assembly state as compared with WT FtsA (17, 34).In this study, we use an in vitro system with purified FtsZ and a purified tagged version of FtsA* to elucidate the role of FtsA in activating constriction of the Z ring in vivo. We show that FtsA*, at physiological concentrations in the presence of ATP or ADP, has significant effects on the assembly of FtsZ filaments.     

The interactions of cell division protein FtsZ with guanine nucleotides

Prokaryotic cell division protein FtsZ, an assembling GTPase, directs the formation of the septosome between daughter cells. FtsZ is an attractive target for the development of new antibiotics. Assembly dynamics of FtsZ is regulated by the binding, hydrolysis, and exchange of GTP. We have determined the energetics of nucleotide binding to model apoFtsZ from Methanococcus jannaschii and studied the kinetics of 2'/3'-O-(N-methylanthraniloyl) (mant)-nucleotide binding and dissociation from FtsZ polymers, employing calorimetric, fluorescence, and stopped-flow methods. FtsZ binds GTP and GDP with K(b) values ranging from 20 to 300 microm(-1) under various conditions. GTP.Mg(2+) and GDP.Mg(2+) bind with slightly reduced affinity. Bound GTP and the coordinated Mg(2+) ion play a minor structural role in FtsZ monomers, but Mg(2+)-assisted GTP hydrolysis triggers polymer disassembly. Mant-GTP binds and dissociates quickly from FtsZ monomers, with approximately 10-fold lower affinity than GTP. Mant-GTP displacement measured by fluorescence anisotropy provides a method to test the binding of any competing molecules to the FtsZ nucleotide site. Mant-GTP is very slowly hydrolyzed and remains exchangeable in FtsZ polymers, but it becomes kinetically stabilized, with a 30-fold slower k(+) and approximately 500-fold slower k(-) than in monomers. The mant-GTP dissociation rate from FtsZ polymers is comparable with the GTP hydrolysis turnover and with the reported subunit turnover in Escherichia coli FtsZ polymers. Although FtsZ polymers can exchange nucleotide, unlike its eukaryotic structural homologue tubulin, GDP dissociation may be slow enough for polymer disassembly to take place first, resulting in FtsZ polymers cycling with GTP hydrolysis similarly to microtubules.     

Response to Therapeutic Plasma Exchange as a Rescue Treatment in Clinically Isolated Syndromes and Acute Worsening of Multiple Sclerosis: A Retrospective Analysis of 90 Patients

Objectives

Experience with therapeutic plasma exchange (TPE) for acute relapses in clinically isolated syndrome (CIS) or multiple sclerosis (MS) patients has been derived from small and inhomogeneous patient populations so far. In the present study, we retrospectively evaluated features associated with TPE response in a larger cohort of CIS and MS patients with acute worsening of disease.

Participants

Ninety CIS and MS patients with acute relapses or acute worsening of symptoms were firstly treated with TPE. The population consisted of 62 women and 28 men with a median age of 38 years (range 18–69 years).

Outcome Measures

Primary endpoint was the clinical response to TPE, focused on the functional improvement of the target neurologic deficit. Secondary endpoint was an improvement in expanded disability status scale (EDSS) scoring.

Results

A clinical response to TPE was observed in 65 out of 90 patients (72.2%), with marked improvement in 18 (20.0%) and moderate improvement in 47 out of 90 patients (52.2%). The median EDSS was reduced from 3.75 before to 3.0 after TPE (p = 0.001). Response to TPE was significantly more frequent in patients with relapsing courses of disease (CIS, RR-MS, p = 0.001), no disease modifying drugs (p = 0.017), gadolinium-positive (Gd⁺) MRI lesions (p = 0.001) and EDSS ≤ 5.0 before TPE (p = 0.014). In the multiple logistic regression analysis only the detection of Gd⁺ MRI lesions was significantly altered (p = 0.004).

Conclusion

Clinical response to TPE was achieved in the majority of our patients. We identified clinical and diagnostic features in CIS and MS relapses that might be helpful to identify patients responding to TPE. Gd⁺ MRI lesions before treatment were the best predictor of the response to TPE in our cohort.     

A New Equation to Estimate Muscle Mass from Creatinine and Cystatin C

Background

With evaluation for physical performance, measuring muscle mass is an important step in detecting sarcopenia. However, there are no methods to estimate muscle mass from blood sampling.

Methods

To develop a new equation to estimate total-body muscle mass with serum creatinine and cystatin C level, we designed a cross-sectional study with separate derivation and validation cohorts. Total body muscle mass and fat mass were measured using dual-energy x-ray absorptiometry (DXA) in 214 adults aged 25 to 84 years who underwent physical checkups from 2010 to 2013 in a single tertiary hospital. Serum creatinine and cystatin C levels were also examined.

Results

Serum creatinine was correlated with muscle mass (P < .001), and serum cystatin C was correlated with body fat mass (P < .001) after adjusting glomerular filtration rate (GFR). After eliminating GFR, an equation to estimate total-body muscle mass was generated and coefficients were calculated in the derivation cohort. There was an agreement between muscle mass calculated by the novel equation and measured by DXA in both the derivation and validation cohort (P < .001, adjusted R² = 0.829, β = 0.95, P < .001, adjusted R² = 0.856, β = 1.03, respectively).

Conclusion

The new equation based on serum creatinine and cystatin C levels can be used to estimate total-body muscle mass.     

By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+

Introduction

The possible role of UCP2 in modulating mitochondrial Ca²⁺-uptake (mCa²⁺-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial.

Methods

Thus, we analyzed mCa²⁺-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca²⁺-transients from mitochondrial ((Ca²⁺)m) and intracellular compartment ((Ca²⁺)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2^-/- mice.

Results

Isolated mitochondria showed a Ru360 sensitive mCa²⁺-uptake, which was significantly decreased in UCP2^-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca²⁺-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2^-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2^-/- cardiomyocytes (Ca²⁺)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca²⁺)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca²⁺-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa²⁺-uptake in WT mitoplasts and (Ca²⁺)m of cardiomyocytes leading to an increase of (Ca²⁺)c while no ATP dependent effect was observed in UCP2^-/-.

Conclusion

Our results indicate regulatory effects of UCP2 on mCa²⁺-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling.     

Slow polymerization of Mycobacterium tuberculosis FtsZ

The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coli and purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90 degrees light scattering. The mycobacterial protein reaches maximum polymerization much more slowly ( approximately 10 min) than E. coli FtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg(2+) and GTP. The minimum concentration of FtsZ needed for polymerization is 3 microM. Electron microscopy shows that polymerized M. tuberculosis FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments. Ethyl 6-amino-2, 3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate+ ++ (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits M. tuberculosis FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.     

Using an Adenosine Triphosphate Bioluminescent Assay to Determine Effective Antibiotic Combinations against Carbapenem-Resistant Gram Negative Bacteria within 24 Hours

Background

Current in vitro combination testing methods involve enumeration by bacterial plating, which is labor-intensive and time-consuming. Measurement of bioluminescence, released when bacterial adenosine triphosphate binds to firefly luciferin-luciferase, has been proposed as a surrogate for bacterial counts. We developed an ATP bioluminescent combination testing assay with a rapid turnaround time of 24h to determine effective antibiotic combinations.

Methods

100 strains of carbapenem-resistant (CR) GNB [30 Acinetobacter baumannii (AB), 30 Pseudomonas aeruginosa (PA) and 40 Klebsiella pneumoniae (KP)] were used. Bacterial suspensions (10⁵ CFU/ml) were added to 96-well plates containing clinically achievable concentrations of multiple single and two-antibiotic combinations. At 24h, the luminescence intensity of each well was measured. Receiver operator characteristic curves were plotted to determine optimal luminescence threshold (T_RLU) to discriminate between inhibitory/non-inhibitory combinations when compared to viable plating. The unweighted accuracy (UA) [(sensitivity + specificity)/2] of T_RLU values was determined. External validation was further done using 50 additional CR-GNB.

Results

Predictive accuracies of T_RLU were high for when all antibiotic combinations and species were collectively analyzed (T_RLU = 0.81, UA = 89%). When individual thresholds for each species were determined, UA remained high. Predictive accuracy was highest for KP (T_RLU = 0.81, UA = 91%), and lowest for AB (T_RLU = 0.83, UA = 87%). Upon external validation, high overall accuracy (91%) was observed. The assay distinguished inhibitory/non-inhibitory combinations with UA of 80%, 94% and 93% for AB, PA and KP respectively.

Conclusion

We developed an assay that is robust at identifying useful combinations with a rapid turn-around time of 24h, and may be employed to guide the timely selection of effective antibiotic combinations.