47 research outputs found
Isolation of native plasma membrane H<sup>+</sup>-ATPase (Pma1p) in both the active and basal activation states
The yeast plasma membrane H(+)-ATPase Pma1p is a P-type ATPase that energizes the yeast plasma membrane. Pma1p exists in two activation states: an autoinhibited basal state and an activated state. Here we show that functional and stable Pma1p can be purified in native form and reconstituted in artificial liposomes without altering its activation state. Acetylated tubulin has previously been reported to maintain Pma1p in the basal state but, as this protein was absent from the purified preparations, it cannot be an essential component of the autoinhibitory mechanism. Purification of and reconstitution of native Pma1p in both activation states opens up for a direct comparison of the transport properties of these states, which allowed us to confirm that the basal state has a low coupling ratio between ATP hydrolysis and protons pumped, whereas the activated state has a high coupling ratio. The ability to prepare native Pma1p in both activation states will facilitate further structural and biochemical studies examining the mechanism by which plasma membrane H(+)-ATPases are autoinhibited
Interaction of plant cell signaling molecules, salicylic acid and jasmonic acid, with the mitochondria of Helicoverpa armigera
Structure and mechanism of Zn^(2+)- transporting P-type ATPases
Zinc is an essential micronutrient for all living organisms. It is required for signalling and proper functioning of a range of proteins involved in, for example, DNA binding and enzymatic catalysis. In prokaryotes and photosynthetic eukaryotes, Zn2+-transporting P-type ATPases of class IB (ZntA) are crucial for cellular redistribution and detoxification of Zn2+ and related elements. Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2·P_i) of ZntA from Shigella sonnei, determined at 3.2 Å and 2.7 Å resolution, respectively. The structures reveal a similar fold to Cu^+-ATPases, with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn^(2+) ions by the transporter. The E2P structure displays a wide extracellular release pathway reaching the invariant residues at the high-affinity site, including C392, C394 and D714. The pathway closes in the E2·P_i state, in which D714 interacts with the conserved residue K693, which possibly stimulates Zn^(2+) release as a built-in counter ion, as has been proposed for H^+-ATPases. Indeed, transport studies in liposomes provide experimental support for ZntA activity without counter transport. These findings suggest a mechanistic link between P_(IB)-type Zn^(2+)-ATPases and P_(III)-type H^+-ATPases and at the same time show structural features of the extracellular release pathway that resemble P_(II)-type ATPases such as the sarcoplasmic/endoplasmic reticulum Ca^(2+)-ATPase (SERCA) and Na^+, K^+-ATPase. These findings considerably increase our understanding of zinc transport in cells and represent new possibilities for biotechnology and biomedicine
Evaluation of appendicitis risk prediction models in adults with suspected appendicitis
Background
Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis.
Methods
A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis).
Results
Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent).
Conclusion
Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified
Roles of plasma membrane proton ATPases AHA2 and AHA7 in normal growth of roots and root hairs in Arabidopsis thaliana
Regulation of renal proximal tubule Na-K-ATPase by prostaglandins
Prostaglandins (PGs) play a number of roles in the kidney, including regulation of salt and water reabsorption. In this report, evidence was obtained for stimulatory effects of PGs on Na-K-ATPase in primary cultures of rabbit renal proximal tubule (RPT) cells. The results of our real-time PCR studies indicate that in primary RPTs the effects of PGE2, the major renal PG, are mediated by four classes of PGE (EP) receptors. The role of these EP receptors in the regulation of Na-K-ATPase was examined at the transcriptional level. Na-K-ATPase consists of a catalytic α-subunit encoded by the ATP1A1 gene, as well as a β-subunit encoded by the ATP1B1 gene. Transient transfection studies conducted with pHβ1-1141 Luc, a human ATP1B1 promoter/luciferase construct, indicate that both PGE1 and PGE2 are stimulatory. The evidence for the involvement of both the cAMP and Ca2+ signaling pathways includes the inhibitory effects of the myristolylated PKA inhibitor PKI, the adenylate cyclase (AC) inhibitor SQ22536, and the PKC inhibitors Gö 6976 and Ro-32-0432 on the PGE1 stimulation. Other effectors that similarly act through cAMP and PKC were also stimulatory to transcription, including norepinephrine and dopamine. In addition to its effects on transcription, a chronic incubation with PGE1 was observed to result in an increase in Na-K-ATPase mRNA levels as well as an increase in Na-K-ATPase activity. An acute stimulatory effect of PGE1 on Na-K-ATPase was observed and was associated with an increase in the level of Na-K-ATPase in the basolateral membrane