Qualitative study of patient experiences of mental distress during TB investigation and treatment in Zambia.

BACKGROUND: The mental health and TB syndemic is a topic that remains under-researched with a significant gap in acknowledging and recognizing patient experiences, particularly in the sub-Saharan African region. In this qualitative study conducted in Zambia, we aimed to explore the lived mental health experiences of TB patients focusing on their multi-layered drivers of distress, and by so doing highlighting contextual factors that influence mental distress in TB patients in this setting. METHODS: The study draws on qualitative data collected in 2018 as part of the Tuberculosis Reduction through Expanded Antiretroviral Treatment and Screening for active TB trial (TREATS) being conducted in Zambia. The data was collected through in-depth interviews with former TB patients (n = 80) from 8 urban communities participating in the TREATS trial. Thematic analysis was conducted. Additional quantitative exploratory analysis mapping mental distress symptoms on demographic, social, economic and TB characteristics of participants was conducted. RESULTS: Most participants (76%) shared that they had experienced some form of mental distress during their TB investigation and treatment period. The reported symptoms ranged in severity. Some participants reported mild distress that did not disrupt their daily lives or ability to adhere to their TB medication, while other participants reported more severe symptoms of distress, for example, 15% of participants shared that they had suicidal ideation and thoughts of self-harm during their time on treatment. Mental distress was driven by unique interactions between individual, social and health level factors most of which were inextricably linked to poverty. Mental distress caused by individual level drivers such as TB morbidity often abated once participants started feeling better, however social, economic and health system level drivers of distress persisted during and beyond TB treatment. CONCLUSION: The findings illustrate that mental distress during TB is driven by multi-layered and intersecting stresses, with the economic stress of poverty often being the most powerful driver. Measures are urgently needed to support TB patients during the investigation and treatment phase, including increased availability of mental health services, better social security safety nets during TB treatment, and interventions targeting TB, HIV and mental health stigma. Trial registration ClinicalTrials.gov NCT03739736 . Trial registration date: November 14, 2018.

Conceptualization, detection, and management of psychological distress and mental health conditions among people with tuberculosis in Zambia: a qualitative study with stakeholders' and TB health workers.

BACKGROUND: In recent years, there has been increased recognition of the need to integrate mental health services into routine tuberculosis (TB) care. For successful integration, policymakers need to first understand the practices of TB health workers in the management of mental health conditions, including depression, anxiety, and psychological distress, and use this to decide how best mental health services could be delivered in tandem with TB services. In this qualitative study we aimed to understand how TB health workers and other stakeholders viewed mental health conditions linked to TB and how they screened and treated these in their patients. METHODS: The study draws on qualitative data collected in 2018 as part of the Tuberculosis Reduction through Expanded Antiretroviral Treatment and Screening for active TB trial (TREATS), conducted in eight urban communities in Zambia. Data were collected through 17 focus group discussions with local health committee members (n = 96) and TB stakeholders (n = 57) present in the communities. Further in-depth interviews were held with key TB health workers (n = 9). Thematic analysis was conducted. RESULTS: TB stakeholders and health workers had an inadequate understanding of mental health and commonly described mental health conditions among TB patients by using stigmatizing terminology and overtones, for example "madness", which often implied a characterological flaw rather an actual illness. Psychological distress was also described as "overthinking", which participants attributed to psychosocial stressors, and was not perceived as a condition that would benefit from mental health intervention. There were no standard screening and treatment options for mental health conditions in TB patients and most TB health workers had no mental health training. TB Stakeholders and health workers understood the negative implications of mental health conditions on TB treatment adherence and overall wellbeing for TB patients. CONCLUSIONS: TB stakeholders and health workers in Zambia have a complex conceptualisation of mental health and illness, that does not support the mental health needs of TB patients. The integration of mental health training in TB services could be beneficial and shift negative attitudes about mental health. Further, TB patients should be screened for mental health conditions and offered treatment. Trial registration number NCT03739736-Registered on the 14th of November 2018- Retrospectively registered- https://clinicaltrials.gov/ct2/results?cond=&term=NCT03739736&cntry=&state=&city=&dist.

Cationic agent contrast-enhanced computed tomography imaging of cartilage correlates with the compressive modulus and coefficient of friction.

OBJECTIVE: The aim of this study is to evaluate whether contrast-enhanced computed tomography (CECT) attenuation, using a cationic contrast agent (CA4+), correlates with the equilibrium compressive modulus (E) and coefficient of friction (µ) of ex vivo bovine articular cartilage. METHODS: Correlations between CECT attenuation and E (Group 1, n = 12) and µ (Group 2, n = 10) were determined using 7 mm diameter bovine osteochondral plugs from the stifle joints of six freshly slaughtered, skeletally mature cows. The equilibrium compressive modulus was measured using a four-step, unconfined, compressive stress-relaxation test, and the coefficients of friction were determined from a torsional friction test. Following mechanical testing, samples were immersed in CA4+, imaged using µCT, rinsed, and analyzed for glycosaminoglycan (GAG) content using the 1,9-dimethylmethylene blue (DMMB) assay. RESULTS: The CECT attenuation was positively correlated with the GAG content of bovine cartilage (R(2) = 0.87, P < 0.0001 for Group 1 and R(2) = 0.74, P = 0.001 for Group 2). Strong and significant positive correlations were observed between E and GAG content (R(2) = 0.90, P < 0.0001) as well as CECT attenuation and E (R(2) = 0.90, P < 0.0001). The CECT attenuation was negatively correlated with the three coefficients of friction: CECT vs µ(static) (R(2) = 0.71, P = 0.002), CECT vs µ(static_equilibrium) (R(2) = 0.79, P < 0.001), and CECT vs µ(kinetic) (R(2) = 0.69, P = 0.003). CONCLUSIONS: CECT with CA4+ is a useful tool for determining the mechanical properties of ex vivo cartilage tissue as the attenuation significantly correlates with the compressive modulus and coefficient of friction.

Contrast agent electrostatic attraction rather than repulsion to glycosaminoglycans affords a greater contrast uptake ratio and improved quantitative CT imaging in cartilage.

PURPOSE: The purpose of this study is to evaluate the effect of contrast agent charge on the contrast agent uptake ratio (CUR) in cartilage and to image the naturally occurring variations in glycosaminoglycan (GAG) content present in bovine articular cartilage. METHODS: In an ex vivo bovine osteochondral plug model, we utilized three charged contrast agents (Gadopentetate/Magnevist [-2], Ioxaglate/Hexabrix [-1], and CA4+ [+4]) and µCT to image cartilage. The X-ray attenuation of the cartilage tissue after equilibration in each contrast agent was also related to the initial X-ray attenuation of each contrast agent in solution to compute the uptake of the respective contrast agent (i.e., the CUR). RESULTS: Use of the cationic contrast agent resulted in significantly higher equilibrium X-ray attenuations in cartilage ECM than either of the anionic contrast agents (Gadopentetate [-2] and Ioxaglate [-1]). The CUR (Mean±SD) as computed in this study was 2.38 (±0.26) for the cationic contrast agent indicating a 2.38 fold increase in computed tomography (CT) attenuation of the cartilage. For the anionic contrast agents, the CUR was 0.62 (±0.26) for Ioxaglate [-1] and 0.52 (±0.17) for Gadopentetate [-2], indicating exclusion of 38% Ioxaglate and 48% Gadopentetate from the cartilage extracellular matrix. The cationic contrast agent exhibited significant correlations between CT attenuation and GAG content whereas Ioxaglate and Gadopentetate did not (R(2)=0.83 for CA4+, R(2)=0.20 for Ioxaglate, and R(2)=0.22 for Gadopentetate). CONCLUSION: Electrostatic attraction of CA4+ allowed effective imaging of the GAG components of articular cartilage at 50% lower molar concentration than Ioxaglate and 20-fold lower molar concentration than Gadopentetate. The CA4+ contrast agent exhibited a significant correlation between CT attenuation and GAG content in ex vivo bovine osteochondral plugs.

A prospective study of psychological distress among mothers of children admitted to a nutritional rehabilitation unit in Malawi.

OBJECTIVES: Accompanying guardians (usually the mother) have a pivotal role in promoting recovery from childhood severe acute malnutrition on Nutritional Rehabilitation Units (NRUs). We describe the prevalence of maternal distress at an NRU in Malawi and identify factors associated with this. We tested the hypothesis that maternal distress during admission would be associated with reduced child weight gain over the 4-week post-discharge period. METHODS: Maternal distress was measured using the Self Reporting Questionnaire (SRQ) administered to mothers of consecutive children during NRU admission. Repeat SRQ was administered to mothers attending a follow-up clinic 4 weeks post discharge. Maternal, child and psychosocial variables were also measured. Child weight change from discharge to follow-up was compared between children of mothers scoring SRQ ≥ 8 and those scoring SRQ < 8. FINDINGS: A total of 244 mothers and their children were recruited. In total, 71% of mothers scored SRQ ≥ 8 during admission. In all, 155 of 222 mothers eligible to complete repeat SRQ did so, and 33.5% scored SRQ ≥ 8. Maternal distress at recruitment was associated with older child age, no confiding relationship with spouse, having had a previous child die, and the child having diarrhoea. Maternal distress at follow-up was associated with older child age, the child having diarrhoea or fever since discharge, and the child being HIV sero-positive. Maternal distress during admission was not associated with child weight gain at 4-week post-discharge follow-up. CONCLUSION: Levels of maternal distress are very high during child admission to an NRU. Persistent distress is associated with child health factors including HIV. Nutritional rehabilitation programmes should pay increased attention to carer psychological wellbeing using targeted evidence-based interventions.

Active site mutations in CheA, the signal-transducing protein kinase of the chemotaxis system in Escherichia coli.

We investigated the functional roles of putative active site residues in Escherichia coli CheA by generating nine site-directed mutants, purifying the mutant proteins, and quantifying the effects of those mutations on autokinase activity and binding affinity for ATP. We designed these mutations to alter key positions in sequence motifs conserved in the protein histidine kinase family, including the N box (H376 and N380), the G1 box (D420 and G422), the F box (F455 and F459), the G2 box (G470, G472, and G474), and the "GT block" (T499), a motif identified by comparison of CheA to members of the GHL family of ATPases. Four of the mutant CheA proteins exhibited no detectable autokinase activity (Kin(-)). Of these, three (N380D, D420N, and G422A) exhibited moderate decreases in their affinities for ATP in the presence or absence of Mg(2+). The other Kin(-) mutant (G470A/G472A/G474A) exhibited wild-type affinity for ATP in the absence of Mg(2+), but reduced affinity (relative to that of wild-type CheA) in the presence of Mg(2+). The other five mutants (Kin(+)) autophosphorylated at rates slower than that exhibited by wild-type CheA. Of these, three mutants (H376Q, D420E, and F455Y/F459Y) exhibited severely reduced k(cat) values, but preserved K(M)(ATP) and K(d)(ATP) values close to those of wild-type CheA. Two mutants (T499S and T499A) exhibited only small effects on k(cat) and K(M)(ATP). Overall, these results suggest that conserved residues in the N box, G1 box, G2 box, and F box contribute to the ATP binding site and autokinase active site in CheA, while the GT block makes little, if any, contribution. We discuss the effects of specific mutations in relation to the three-dimensional structure of CheA and to binding interactions that contribute to the stability of the complex between CheA and Mg(2+)-bound ATP in both the ground state and the transition state for the CheA autophosphorylation reaction.

Recent research on alcohol tolerance and dependence.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Hiroshi Suwaki and Harold Kalant. The presentations were (1) Influence of ADH genotypes on acute alcohol withdrawal syndrome in Japanese, by Susumu Higuchi; (2) Use of genetic analyses to refine phenotypes related to alcohol tolerance and dependence, by John C. Crabbe; (3) Neurochemical basis for alcohol dependence, by Seitaro Ohkuma and Masashi Katsura; (4) Adenylyl cyclase and development of tolerance to addictive drugs, by Masami Yoshimura; (5) Tolerance in rat lines selectively bred for alcohol preference, by Robert C. Stewart and Ting-Kai Li; and (6) Ethanol reinforcement, dependence, and vulnerability to relapse: Interactions between neuroadaptive and conditioning factors, by Friedbert Weiss.

Rapid phosphotransfer to CheY from a CheA protein lacking the CheY-binding domain.

The histidine protein kinase CheA plays a central role in the bacterial chemotaxis signal transduction pathway. Autophosphorylated CheA passes its phosphoryl group to CheY very rapidly (k(cat) approximately 750 s(-)(1)). Phospho-CheY in turn influences the direction of flagellar rotation. The autophosphorylation site of CheA (His(48)) resides in its N-terminal P1 domain. The adjacent P2 domain provides a high-affinity binding site for CheY, which might facilitate the phosphotransfer reaction by tethering CheY in close proximity to the phosphodonor located in P1. To explore the contribution of P2 to the CheA --> CheY phosphotransfer reaction in the Escherichia coli chemotaxis system, we examined the transfer kinetics of a mutant CheA protein (CheADeltaP2) in which the 98 amino acid P2 domain had been replaced with an 11 amino acid linker. We used rapid-quench and stopped-flow fluorescence experiments to monitor phosphotransfer to CheY from phosphorylated wild-type CheA and from phosphorylated CheADeltaP2. The CheADeltaP2 reaction rates were significantly slower and the K(m) value was markedly higher than the corresponding values for wild-type CheA. These results indicate that binding of CheY to the P2 domain of CheA indeed contributes to the rapid kinetics of phosphotransfer. Although phosphotransfer was slower with CheADeltaP2 (k(cat)/K(m) approximately 1.5 x 10(6) M(-)(1) s(-)(1)) than with wild-type CheA (k(cat)/K(m) approximately 10(8) M(-)(1) s(-)(1)), it was still orders of magnitude faster than the kinetics of CheY phosphorylation by phosphoimidazole and other small molecule phosphodonors (k(cat)/K(m) approximately 5-50 M(-)(1) s(-)(1)). We conclude that the P1 domain of CheA also makes significant contributions to phosphotransfer rates in chemotactic signaling.

Kinetic characterization of CheY phosphorylation reactions: comparison of P-CheA and small-molecule phosphodonors.

In the chemotaxis system of Escherichia coli, phosphorylation of the CheY protein plays an important role in regulating the swimming pattern of the cell. In vitro, CheY can be phosphorylated either by phosphotransfer from phospho-CheA or by acquiring a phosphoryl group from any of a variety of small, high-energy phosphodonor molecules such as acetyl phosphate. Previous work explored the rapid kinetics of CheY phosphorylation by CheA. Here we extend that work and examine the kinetics of CheY phosphorylation by several small-molecule phosphodonors, including acetyl phosphate, benzoyl phosphate, carbamoyl phosphate, 2-methoxybenzoyl phosphate, and phosphoramidate. Our results indicate that these phosphodonors bind to CheY with relatively low affinity (Ks values ranging from 10 to 600 mM) and that the rate constant (kphos) for phosphotransfer at saturating phosphodonor concentrations is relatively slow (values ranging from 0.05 to 0.5 s-1). By contrast, under identical conditions, phosphorylation of CheY by phospho-CheA occurs much more rapidly (kphos approximately 800 s-1) and reflects CheY binding to phospho-CheA considerably more tightly (Ks approximately 60 microM) than it does to the small-molecule phosphodonors. In comparing CheA-mediated phosphorylation of CheY to small-molecule-mediated phosphorylation of CheY, the large difference in kphos values suggests that phospho-CheA makes significant contributions to the catalysis of CheY phosphorylation. The effects of pH and ionic strength on CheY phosphorylation kinetics were also investigated. For CheA-->CheY phosphotransfer, increasing ionic strength resulted in increased Ks values while kphos was unaffected. For CheY phosphorylation by small-molecule phosphodonors, increasing ionic strength resulted in decreasing Ks values and increasing kphos values. The significance of these effects is discussed in relation to the catalytic mechanism of CheY phosphorylation by phospho-CheA and small-molecule phosphodonors.

Yeast Skn7p activity is modulated by the Sln1p-Ypd1p osmosensor and contributes to regulation of the HOG pathway.

Activation and control of the yeast HOG (High Osmolarity Glycerol) MAP kinase cascade is accomplished, in part, by a two-component sensory-response circuit comprised of the osmosensing histidine protein kinase Sln1p, the phospho-relay protein Ypd1p, and the response regulator protein Ssk1p. We found that deletion of SLN1 and/or YPD1 reduces reporter gene transcription driven by a second two-component response regulator -- Skn7p. The effect of sln1delta and ypd1delta mutations upon Skn7p activity is dependent on a functional two-component phosphorylation site (D427) in Skn7p, suggesting that Sln1p and Ypd1p may act as phosphodonors for Skn7p. We also observed that loss of PTC1 (a protein serine/threonine phosphatase implicated in negative control of the HOG pathway) in a skn7delta background results in severely retarded growth and in morphological defects. Deletion of either PBS2 or HOG1 alleviates the slow growth phenotype of ptc1delta skn7delta cells, suggesting that Skn7p may participate, in concert with known regulatory components, in modulating HOG pathway activity. The contribution of Skn7p to HOG pathway regulation appears to be modulated by the receiver domain, since non-phosphorylatable Skn7pD427N is unable to fully restore growth to ptc1/skn7 cells.

TNP-ATP and TNP-ADP as probes of the nucleotide binding site of CheA, the histidine protein kinase in the chemotaxis signal transduction pathway of Escherichia coli.

The interaction of CheA with ATP has important consequences in the chemotaxis signal transduction pathway of Escherichia coli. This interaction results in autophosphorylation of CheA, a histidine protein kinase. Autophosphorylation of CheA sets in motion a chain of biochemical events that enables the chemotaxis receptor proteins to communicate with the flagellar motors. As a result of this communication, CheA allows the receptors to control the cell swimming pattern in response to gradients of attractant and repellent chemicals. To probe CheA interactions with ATP, we investigated the interaction of CheA with the fluorescent nucleotide analogues TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate] and TNP-ADP. Spectroscopic studies indicated that CheA bound TNP-ATP and TNP-ADP with high affinity (micromolar Kd values) and caused a marked enhancement of the fluorescence of the TNP moiety of these modified nucleotides. Analysis of titration experiments indicated a binding stoichiometry of two molecules of TNP-ATP (TNP-ADP) per CheA dimer and suggested that the two binding sites on the CheA dimer operate independently. Binding of TNP-ATP to CheA was inhibited by ATP, and analysis of this inhibition indicated that the CheA dimer binds 2 molecules of ATP. Competition experiments also indicated that CheA binds TNP-ATP considerably more tightly than it binds unmodified ATP. Binding of TNP-ADP to CheA was inhibited by ADP in a similar manner. TNP-ATP was not a substrate for CheA and served as a potent inhibitor of CheA autophosphorylation (Ki < 1 microM). The glycine-rich regions (G1 and G2) of CheA and other histidine protein kinases have been presumed to play important roles in ATP binding and/or catalysis of CheA autophosphorylation, although few experimental tests of these functional assignments have been made. Here, we demonstrate that a CheA mutant protein with Gly-->Ala substitutions in G1 and G2 has a markedly reduced affinity for ATP and ADP, as measured by Hummel-Dreyer chromatography. This mutant protein also bound TNP-ATP and TNP-ADP very poorly and had no detectable autokinase activity. Surprisingly, a distinct single-site substitution in G2 (Gly470-->Lys) had no observable effect on the affinity of CheA for ATP and ADP, despite the fact that it rendered CheA completely inactive as an autokinase. This mutant protein also bound TNP-ATP and TNP-ADP with affinities and stoichiometries that were indistinguishable from those observed with wild-type CheA. These results provide some preliminary insight into the possible functional roles of G1 and G2, and they suggest that TNP-nucleotides are useful tools for exploring the effects of additional mutations on the active site of CheA.

Kinetic characterization of phosphotransfer between CheA and CheY in the bacterial chemotaxis signal transduction pathway.

Phosphorylation of the CheY protein is a crucial step in the chemotaxis signal transduction pathway of Escherichia coli. CheY becomes phosphorylated by acquiring a phosphoryl group from CheA, an autophosphorylating protein kinase. In this study, we utilized a rapid-quench instrument to investigate the kinetics of phosphotransfer in single-turnover experiments. Our results are consistent with a three-step mechanism for the CheA-to-CheY phosphotransfer reaction: (i) reversible binding of CheY to P-CheA; (ii) rapid, reversible phosphotransfer to CheY; (iii) reversible dissociation of the resulting CheA x CheY-P complex. Investigation of the effect of CheY concentration on the observed rate of phosphotransfer demonstrated saturation kinetics; the extrapolated limiting rate constant for phosphotransfer was 650 +/- 200 s(-1), while the Km value indicated from this work was 6.5 +/- 2 microM. We demonstrated that the CheA-CheY phosphotransfer reaction was reversible by observing partial transfer of [32P]phosphate from CheY-P to CheA and by observing the effect of high concentrations of unphosphorylated CheA on the equilibrium: P-CheA + CheY <--> CheA + CheY-P. We found that the rate of phosphotransfer from P-CheA to CheY can be inhibited by unphosphorylated CheA as well as by a fragment of CheA (CheA124-257) that contains the CheY binding site; these results suggest that the unphosphorylated form of CheA can effectively compete with P-CheA for available CheY (Kd approximately 1.5 +/- 0.6 microM for the CheY x CheA124-257 complex and for the CheY x CheA complex).

Identification of ASK10 as a multicopy activator of Skn7p-dependent transcription of a HIS3 reporter gene.

Recent evidence has demonstrated that the yeast Skn7p appears to act as a 'response regulator' in a eukaryotic 'two-component' signal transduction pathway. A search to identify possible regulators of the SKN7 mediated 'two-component' regulatory system has identified Ask10p as a novel potential transcription factor. The ASK10 sequence has been deposited in GenBank with Accession Number U27209.

Yeast Skn7p functions in a eukaryotic two-component regulatory pathway.

Previous analysis of the amino acid sequence of Skn7p, the product of the yeast SKN7 gene, revealed a potential 'receiver motif' homologous to that found in bacterial response regulators (signal-transducing effector proteins regulated by phosphorylation at a conserved aspartate residue corresponding to position D427 in Skn7p). We determined the effects of D427N and D427E mutations in Skn7p. The D427N substitution resulted in diminished activity in four independent in vivo assays of Skn7p function, while the D427E mutation enhanced Skn7p activity in these assays. Our results are consistent with predictions based on the bacterial two-component paradigm and provide experimental evidence that a receiver motif functions in regulating the activity of Skn7p in a eukaryote. Skn7p suppressed growth defects associated with a pkc1 delta mutation, raising the possibility that PKC1 might play a role in regulating Skn7p. However, epistasis experiments indicate that Skn7p does not appear to function directly downstream of the PKC1-MAP kinase pathway. Rather, Skn7p may function in a two-component signal transduction pathway that acts in parallel with the PKC1 cascade to regulate growth at the cell surface. We present evidence suggesting that Skn7p serves as a transcription factor in such a signaling pathway.

The short form of CheA couples chemoreception to CheA phosphorylation.

Escherichia coli cells express two forms of the chemotaxis-associated CheA protein, CheAL and CheAS, as the result of translational initiation at two distinct in-frame initiation sites in the gene cheA. The long form, CheAL, plays a crucial role in chemotactic signal transduction. As a histidine protein kinase, it first autophosphorylates at amino acid His-48; then, it phosphorylates two other chemotaxis proteins, CheY and CheB. The short form, CheAS, lacks the amino-terminal 97 amino acids of CheAL and, therefore, does not contain the site of autophosphorylation. However, it does retain a functional kinase domain. As a consequence, CheAS can mediate transphosphorylation of kinase-deficient CheAL variants. Here we demonstrate in vitro that CheAS also can mediate transphosphorylation of a CheAL variant that lacks the C-terminal segment, a portion of the protein which is thought to interact with CheW and the chemoreceptors. The presence of CheW and the chemoreceptor Tsr enhances this activity and results in modulation of the transphosphorylation rate in response to the Tsr ligand, L-serine. Because CheAS can mediate this activity, it can restore chemotactic ability to Escherichia coli cells that express this truncated CheAL variant.

pH dependence of CheA autophosphorylation in Escherichia coli.

Chemotaxis by cells of Escherichia coli and Salmonella typhimurium depends upon the ability of chemoreceptors called transducers to communicate with switch components of flagellar motors to modulate swimming behavior. This communication requires an excitatory pathway composed of the cytoplasmic signal transduction proteins, CheAL, CheAS, CheW, CheY, and CheZ. Of these, the autokinase CheAL is most central. Modifications or mutations that affect the rate at which CheAL autophosphorylates result in profound chemotactic defects. Here we demonstrate that pH can affect CheAL autokinase activity in vitro. This activity exhibits a bell-shaped dependence upon pH within the range 6.5 to 10.0, consistent with the notion that two proton dissociation events affect CheAL autophosphorylation kinetics: one characterized by a pKa of about 8.1 and another exhibiting a pKa of about 8.9. These in vitro results predict a decrease in the rate of CheAL autophosphorylation in response to a reduction in intracellular pH, a decrease that should cause increased counterclockwise flagellar rotation. We observed such a response in vivo for cells containing a partially reconstituted chemotaxis system. Benzoate (10 mM, pH 7.0), a weak acid that when undissociated readily traverses the cytoplasmic membrane, causes a reduction of cytoplasmic pH from 7.6 to 7.3. In response to this reduction, cells expressing CheAL, CheAS, and CheY, but not transducers, exhibited a small but reproducible increase in the fraction of time that they spun their flagellar motors counterclockwise. The added presence of CheW and the transducers Tar and Trg resulted in a more dramatic response. The significance of our in vitro results, their relationships to regulation of swimming behavior, and the mechanisms by which transducers might affect the pH dependence of CheA autokinase activity are discussed.

Mutational activation of CheA, the protein kinase in the chemotaxis system of Escherichia coli.

In Escherichia coli and Salmonella typhimurium, appropriate changes of cell swimming patterns are mediated by CheA, an autophosphorylating histidine protein kinase whose activity is regulated by receptor/transducer proteins. The molecular mechanism underlying this regulation remains unelucidated but may involve CheA shifting between high-activity and low-activity conformations. We devised an in vivo screen to search for potential hyperkinase variants of CheA and used this screen to identify two cheA point mutations that cause the CheA protein to have elevated autokinase activity. Each point mutation resulted in alteration of proline 337. In vitro, CheA337PL and CheA337PS autophosphorylated significantly more rapidly than did wild-type CheA. This rate enhancement reflected the higher affinities of the mutant proteins for ATP and an increased rate constant for acquisition by CheA of the gamma-phosphoryl group of ATP within a kinetically defined CheA.ATP complex. In addition, the mutant proteins reacted with ADP more rapidly than did wild-type CheA. We considered the possibility that the mutations served to lock CheA into an activated signaling conformation; however, we found that both mutant proteins were regulated in a normal fashion by the transducer Tsr in the presence of CheW. We exploited the activated properties of one of these mutants to investigate whether the CheA subunits within a CheA dimer make equivalent contributions to the mechanism of trans phosphorylation. Our results indicate that CheA trans phosphorylation may involve active-site residues that are located both in cis and in trans to the autophosphorylation site and that the two protomers of a CheA dimer make nonequivalent contributions in determining the affinity of the ATP-binding site(s) of CheA.

Kinetics of CheA autophosphorylation and dephosphorylation reactions.

The protein kinase CheA of Escherichia coli plays a central role in the signal transduction pathway controlling the swimming behavior of the cell in response to extracellular chemical gradients. CheA autophosphorylates at a rate controlled by the ligand binding state of chemotaxis receptor/transducer proteins. CheA directs the activities of CheY and CheB, effector proteins that become phosphorylated as a result of their interaction with phospho-CheA. In this study, we performed a detailed kinetic analysis of CheA's autophosphorylation reaction, and its dephosphorylation by ADP. Our kinetic data are consistent with a three-step mechanism for CheA autophosphorylation/dephosphorylation involving (i) substrate binding, (ii) phospho-transfer, and (iii) product release. We determined the dissociation constant for the kinetically defined CheA.ATP complex to be approximately 300 microM and the limiting rate constant for autophosphorylation to be approximately 0.026 s-1 at saturating ATP concentration. Our results indicate that the apparent dissociation constant of the phospho-CheA.ADP complex is approximately 42 microM and that the limiting rate constant for CheA dephosphorylation is approximately 0.028 s-1 at saturating ADP concentration. We corroborated the kinetically determined Kd values by performing independent ligand binding experiments. In addition, we found that the kinetics of trans-phosphorylation, involving mutant proteins CheA48HQ and CheA470GK, exhibited kinetic properties similar to those observed for autophosphorylation of wild-type CheA, although the limiting rate constant (0.008 s-1) was somewhat slower for this trans-phosphorylation reaction. These results will provide a framework for assessing the effects of various cheA mutations as well as for exploring the nature of CheA regulation by the chemotaxis receptor/transducer proteins.

Pore geometry information via pulsed field gradient NMR.

Studies of echo attenuation at long diffusion times in pulsed field gradient NMR experiments on a variety of rock core samples are interpreted in the light of recent theoretical analysis of the effect of pore geometry and surface relaxation. This study is motivated by the need to test the applicability of that theory to real rock systems.