232 research outputs found
Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective.
The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking
Insights into bacterial lipoprotein trafficking from a structure of LolA bound to the LolC periplasmic domain.
In Gram-negative bacteria, outer-membrane lipoproteins are essential for maintaining cellular integrity, transporting nutrients, establishing infections, and promoting the formation of biofilms. The LolCDE ABC transporter, LolA chaperone, and LolB outer-membrane receptor form an essential system for transporting newly matured lipoproteins from the outer leaflet of the cytoplasmic membrane to the innermost leaflet of the outer membrane. Here, we present a crystal structure of LolA in complex with the periplasmic domain of LolC. The structure reveals how a solvent-exposed β-hairpin loop (termed the "Hook") and trio of surface residues (the "Pad") of LolC are essential for recruiting LolA from the periplasm and priming it to receive lipoproteins. Experiments with purified LolCDE complex demonstrate that association with LolA is independent of nucleotide binding and hydrolysis, and homology models based on the MacB ABC transporter predict that LolA recruitment takes place at a periplasmic site located at least 50 Å from the inner membrane. Implications for the mechanism of lipoprotein extraction and transfer are discussed. The LolA-LolC structure provides atomic details on a key protein interaction within the Lol pathway and constitutes a vital step toward the complete molecular understanding of this important system.This work was supported by grants from the UK Medical Research Council (MR/N000994/1) and the Wellcome Trust (101828/Z/13/Z)
The effects of methylphenidate on cognitive performance of healthy male rats
We aimed to investigate the effects of methylphenidate (MPH) in healthy rats on two distinct radial maze tasks which rely on brain structures and neurotransmitters known to be affected by MPH: the Random Foraging Non-Delay Task (RFNDT) and the Delayed Spatial Win Shift Task (DSWT). Hooded Lister rats were trained to complete either the RFNDT or the DSWT having received oral treatment of either a vehicle or MPH (3.0 mg/kg and 5.0 mg/kg for RFNDT, 3.0 mg/kg for DSWT). We found no effect of MPH on the RFNDT relative to the control group. However, those treated with 5.0 mg/kg MPH did take significantly longer to reach criterion performance than those treated with the 3.0 mg/kg MPH, suggesting some doses of MPH can have detrimental effects. For the DSWT, if MPH was present in both phases, performance did not differ from when it was absent in both phases. However, when present in only one phase there was an increase in errors made, although this only reached significance for when MPH was present only in the test-phase. These data suggest that MPH may have detrimental effects on task performance and can result in state-dependent effects in healthy individuals
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Structure of CYRI-B (FAM49B), a key regulator of cellular actin assembly.
In eukaryotes, numerous fundamental processes are controlled by the WAVE regulatory complex (WRC) that regulates cellular actin polymerization, crucial for cell motility, cell-cell adhesion and epithelial differentiation. Actin assembly is triggered by interaction of the small GTPase Rac1 with CYFIP1, a key component of the WRC. Previously known as FAM49B, CYRI-B is a protein that is highly conserved across the Eukaryota and has recently been revealed to be a key regulator of Rac1 activity. Mutation of CYRI-B or alteration of its expression therefore leads to altered actin nucleation dynamics, with impacts on lamellipodia formation, cell migration and infection by intracellular pathogens. In addition, knockdown of CYRI-B expression in cancer cell lines results in accelerated cell proliferation and invasiveness. Here, the structure of Rhincodon typus (whale shark) CYRI-B is presented, which is the first to be reported of any CYRI family member. Solved by X-ray crystallography, the structure reveals that CYRI-B comprises three distinct α-helical subdomains and is highly structurally related to a conserved domain present in CYFIP proteins. The work presented here establishes a template towards a better understanding of CYRI-B biological function
Health sector involvement in the management of female genital mutilation/cutting in 30 countries
Background: For the last decades, the international community has emphasised the importance of a multisectoral approach to tackle female genital mutilation (FGM/C). While considerable improvement concerning legislations and community involvement is reported, little is known about the involvement of the health sector. Method: A mixed methods approach was employed to map the involvement of the health sector in the management of FGM/C both in countries where FGM/C is a traditional practice (countries of origin), and countries where FGM/C is practiced mainly by migrant populations (countries of migration). Data was collected in 2016 using a pilot-tested questionnaire from 30 countries (11 countries of origin and 19 countries of migration). In 2017, interviews were conducted to check for data accuracy and to request relevant explanations. Qualitative data was used to elucidate the quantitative data. Results: A total of 24 countries had a policy on FGM/C, of which 19 had assigned coordination bodies and 20 had partially or fully implemented the plans. Nevertheless, allocation of funding and incorporation of monitoring and evaluation systems was lacking in 11 and 13 of these countries respectively. The level of the health sectors' involvement varied considerably across and within countries. Systematic training of healthcare providers (HCP) was more prevalent in countries of origin, whereas involvement of HCP in the prevention of FGM/C was more prevalent in countries of migration. Most countries reported to forbid HCP from conducting FGM/C on both minors and adults, but not consistently forbidding re-infibulation. Availability of healthcare services for girls and women with FGM/C related complications also varied between countries dependent on the type of services. Deinfibulation was available in almost all countries, while clitoral reconstruction and psychological and sexual counselling were available predominantly in countries of migration and then in less than half the countries. Finally, systematic recording of FGM/C in medical records was completely lacking in countries of origin and very limited in countries of migration. Conclusion: Substantial progress has been made in the involvement of the health sector in both the treatment and prevention of FGM/C. Still, there are several areas in need for improvement, particularly monitoring and evaluatio
Elasticity Maps of Living Neurons Measured by Combined Fluorescence and Atomic Force Microscopy
Detailed knowledge of mechanical parameters such as cell elasticity,
stiffness of the growth substrate, or traction stresses generated during axonal
extensions is essential for understanding the mechanisms that control neuronal
growth. Here we combine Atomic Force Microscopy based force spectroscopy with
Fluorescence Microscopy to produce systematic, high-resolution elasticity maps
for three different types of live neuronal cells: cortical (embryonic rat),
embryonic chick dorsal root ganglion, and P-19 (mouse embryonic carcinoma stem
cells) neurons. We measure how the stiffness of neurons changes both during
neurite outgrowth and upon disruption of microtubules of the cell. We find
reversible local stiffening of the cell during growth, and show that the
increase in local elastic modulus is primarily due to the formation of
microtubules. We also report that cortical and P-19 neurons have similar
elasticity maps, with elastic moduli in the range 0.1-2 kPa, with typical
average values of 0.4 kPa (P-19) and 0.2 kPa (cortical). In contrast, DRG
neurons are stiffer than P-19 and cortical cells, yielding elastic moduli in
the range 0.1-8 kPa, with typical average values of 0.9 kPa. Finally, we report
no measurable influence of substrate protein coating on cell body elasticity
for the three types of neurons
Structure and mechanotransmission mechanism of the MacB ABC transporter superfamily.
MacB is an ABC transporter that collaborates with the MacA adaptor protein and TolC exit duct to drive efflux of antibiotics and enterotoxin STII out of the bacterial cell. Here we present the structure of ATP-bound MacB and reveal precise molecular details of its mechanism. The MacB transmembrane domain lacks a central cavity through which substrates could be passed, but instead conveys conformational changes from one side of the membrane to the other, a process we term mechanotransmission. Comparison of ATP-bound and nucleotide-free states reveals how reversible dimerization of the nucleotide binding domains drives opening and closing of the MacB periplasmic domains via concerted movements of the second transmembrane segment and major coupling helix. We propose that the assembled tripartite pump acts as a molecular bellows to propel substrates through the TolC exit duct, driven by MacB mechanotransmission. Homologs of MacB that do not form tripartite pumps, but share structural features underpinning mechanotransmission, include the LolCDE lipoprotein trafficking complex and FtsEX cell division signaling protein. The MacB architecture serves as the blueprint for understanding the structure and mechanism of an entire ABC transporter superfamily and the many diverse functions it supports
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