Towards Molecular Understanding of the pH Dependence Characterizing NhaA of Which Structural Fold is Shared by Other Transporters.

Na+/H+ antiporters comprise a super-family (CPA) of membrane proteins that are found in all kingdoms of life and are essential in cellular homeostasis of pH, Na+ and volume. Their activity is strictly dependent on pH, a property that underpins their role in pH homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystal structure provided insight into the architecture of this molecular machine. However, the mechanism of the strict pH dependence of NhaA is missing. Here, as a follow up of a recent evolutionary analysis that identified a 'CPA motif', we rationally designed three E. coli NhaA mutants: D133S, I134T, and the double mutant D133S-I134T. Exploring growth phenotype, transport activity and Li+-binding of the mutants, we revealed that Asp133 does not participate directly in proton binding, nor does it directly dictate the pH-dependent transport of NhaA. Strikingly, the variant I134T lost some of the pH control, and the D133S-Il134T double mutant retained Li+ binding in a pH independent fashion. Concurrent to loss of pH control, these mutants bound Li+ more strongly than the WT. Both positions are in close vicinity to the ion-binding site of the antiporter, attributing the results to electrostatic interaction between these residues and Asp164 of the ion-binding site. This is consistent with pH sensing resulting from direct coupling between cation binding and deprotonation in Asp164, which applies also to other CPA antiporters that are involved in human diseases.

Effect of patient and nurse ethnicity on emergency department analgesia for children with appendicitis in israeli government hospitals.

BACKGROUND: Ethnicity is a risk factor for disparate Emergency Department (ED) analgesia. We aimed to explore ethnic variations in the administration of ED analgesia to children with acute appendicitis in Israeli government hospitals. METHODS: Children discharged with an International Classification of Disease-Ninth Revision diagnosis of acute appendicitis between 2010 and 2015 were included. The association between patient ethnicity (Jewish, Arab) and analgesia administration (any, opioid) was assessed. Age, gender, triage category, pain score and time of arrival were tested as possible confounders. The effect of patient-nurse ethnic discordance (PNED) was examined. RESULTS: Overall, 4714 children with acute appendicitis, 3520 Jewish and 1194 Arab, were cared for in the EDs; 1516 (32.2%) received any analgesia and 368 (7.8%) opioid analgesia. Stratified by pain score, no statistical differences were found in the administration of any or opioid analgesia between Jewish and Arab patients with either severe pain or moderate pain. In multivariate modelling adjusted for pain score and triage category, the rates of any analgesia for Arab and Jewish patients were 31.8% (95% CI, 30.9-32.6) and 36.5% (95% CI, 36.0-36.9), adjusted OR (aOR) = 1.16 (95% CI, 0.98-1.38), respectively. The rates of opioid analgesia for Arab and Jewish patients were 8.5% (95% CI, 8.2-8.9) and 7.9% (95% CI, 7.3-8.7), aOR = 0.77; (95% CI, 0.59-1.22), respectively. Jewish and Arab nurses treated proportionally fewer patients from the opposite ethnicity with any analgesia (p < 0.01). CONCLUSION: Emergency Department analgesia was markedly low, and not associated with patient ethnicity. PNED was associated with decreased rates of analgesia. SIGNIFICANCE: Emergency Department analgesia for children with acute appendicitis in Israeli government hospitals is markedly low. Patient-provider ethnic discordance may negatively influence the provision of analgesia. Significant efforts should be undertaken in order to increase analgesia provision rates and reduce social inequality.

NhaA of Escherichia coli, as a model of a pH-regulated Na+/H+antiporter.

Na(+)/H(+) antiporters are ubiquitous membrane proteins that are involved in homeostasis of H(+) and Na(+) throughout the biological kingdom. Corroborating their role in pH homeostasis, many of the Na(+)/H(+) antiporter proteins are regulated directly by pH. The pH regulation of NhaA, the Escherichia coli Na(+)/H(+) antiporter (EcNhaA), as of other, both eukaryotic and prokaryotic Na(+)/H(+) antiporters, involves a pH sensor and conformational changes in different parts of the protein that transduce the pH signal into a change in activity. Thus, residues that affect the pH response, the translocation or both activities cluster in separate domains along the antiporter molecules. Importantly, in the NhaA family, these domains are conserved. Helix-packing model of EcNhaA based on cross-linking data suggests, that in the three dimensional structure of NhaA, residues that affect the pH response may be in close proximity, forming a single pH sensitive domain. Therefore, it is suggested that, despite considerable differences in the primary structure of the antiporters from the bacterial NhaA to the mammalian NHEs, their three-dimensional architectures are conserved. Test of this possibility awaits the atomic resolution of the 3D structure of the antiporters.

Proximity of cytoplasmic and periplasmic loops in NhaA Na+/H+ antiporter of Escherichia coli as determined by site-directed thiol cross-linking.

The unique trypsin cleavable site of NhaA, the Na(+)/H(+) antiporter of Escherichia coli, was exploited to detect a change in mobility of cross-linked products of NhaA by polyacrylamide gel electrophoresis. Double-Cys replacements were introduced into loops, one on each side of the trypsin cleavage site (Lys 249). The proximity of paired Cys residues was assessed by disulfide cross-linking of the two tryptic fragments, using three homobifunctional cross-linking agents: 1,6-bis(maleimido)hexane (BMH), N,N'-o-phenylenedimaleimide (o-PDM), and N,N'-p-phenylenedimaleimide (p-PDM). The interloop cross-linking was found to be very specific, indicating that the loops are not merely random coils that interact randomly. In the periplasmic side of NhaA, two patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently between the double-Cys replacements A118C/S286C, N177C/S352C, and H225C/S352C; (b) only BMH cross-links the double-Cys replacements A118C/S352C, N177C/S286C, and H225C/S286C. In the cytoplasmic side of NhaA, three patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently the pairs of Cys replacements L4C/E252C, S146C/L316C, S146C/R383C, and E241C/E252C; (b) BMH and p-PDM cross-link efficiently the pairs of Cys replacements S87C/E252C, S87C/L316C, and S146C/E252C; (c) none of the reagents cross-links the double-Cys replacements L4C/L316C, L4C/R383C, S87C/R383C, A202C/E252C, A202C/L316C, A202C/R383C, E241C/L316C, and E241C/R383C. The data reveal that the N-terminus and loop VIII-IX that have previously been shown to change conformation with pH are in close proximity within the NhaA protein. The data also suggest close proximity between N-terminal and C-terminal helices at both the cytoplasmic and the periplasmic face of NhaA.

Oligomerization of NhaA, the Na+/H+ antiporter of Escherichia coli in the membrane and its functional and structural consequences.

Recently, a two-dimensional crystal structure of NhaA, the Na+/H+ antiporter of Escherichia coli has been obtained [Williams, K. A., Kaufer, U. G., Padan, E., Schuldiner, S. and Kühlbrandt, W. (1999) EMBO J., 18, 3558-3563]. In these crystals NhaA exists as a dimer. Using biochemical and genetic approaches here we show that NhaA exists in the native membrane as a homooligomer. Functional complementation between the polypeptides of NhaA was demonstrated by coexpression of pairs of conditional lethal (at high pH in the presence of Na+) mutant alleles of nhaA in EP432, a strain lacking antiporters. Physical interaction in the membrane was shown between the His-tagged NhaA polypeptide which is readily affinity purified from DM-solubilized membranes with a Ni2+-NTA column and another which is not; only when coexpressed did both copurify on the column. The organization of the oligomer in the membrane was studied in situ by site-directed cross-linking experiments. Cysteine residues were introduced--one per NhaA--into certain loops of Cys-less NhaA, so that only intermolecular cross-linking could take place. Different linker-size cross-linkers were applied to the membranes, and the amount of the cross-linked protein was analyzed by mobility shift on SDS-PAGE. The results are consistent with homooligomeric NhaA and the location of residue 254 in the interface between monomers. Intermolecular cross-linking of V254C caused an acidic shift in the pH profile of NhaA.

Continuous measurement of core body temperature in preterm infants.

We tested a transcutaneous core temperature sensor using a method that relies on the principle of zero heat flow. We tested the hypothesis that transcutaneous and rectal temperatures would track within 0.3 degrees C of each other for >90% of the time. A thermistor was placed between the infant's abdomen or back and the incubator's or radiant warmer's mattress, or within the axilla, attached to the skin with a foam adhesive disk insulator. Thirty preterm infants were either placed on their abdomens or backs in a convective incubator or under a radiant warmer, and continuous transcutaneous and rectal temperatures were measured for 1 hour. There were no significant differences between abdominal and core temperatures or between axillary and core temperatures measured in double-walled convective incubators or in radiant warmers. The rectal-abdominal temperature difference was significantly less than the rectal-axillary difference (p < 0.02) in convective incubators, but not when the infant was placed prone under radiant warmers (p = 0.27). Transcutaneous thermometry is reliable for monitoring core body temperature as indicated by rectal temperature in stable preterm infants in a convective incubator.

The monoclonal antibody 1F6 identifies a pH-dependent conformational change in the hydrophilic NH(2) terminus of NhaA Na(+)/H(+) antiporter of Escherichia coli.

One of the most interesting properties of the NhaA Na(+)/H(+) antiporter of Escherichia coli is the strong regulation of its activity by pH. This regulation is accompanied by a conformational change that can be probed by digestion with trypsin and involves the hydrophilic loop connecting the transmembrane helices VIII-IX. In the present work we show that a monoclonal antibody (mAb), 1F6, recognizes yet another domain of NhaA in a pH-dependent manner. This antibody binds NhaA at pH 8.5 but not at pH 4.5, whereas two other mAbs bind to NhaA independently of pH. The epitope of mAb 1F6 was located at the NH(2) terminus of NhaA by probing proteolytic fragments in Western blot analysis and amino acid sequencing. The antibody bound to the peptide HLHRFFSS, starting at the third amino acid of NhaA. A synthetic peptide with this sequence was shown to bind mAb 1F6 both at acidic and alkaline pH suggesting that this peptide is accessible to mAb 1F6 in the native protein only at alkaline pH. Although slightly shifted to acidic pH, the pH profile of the binding of mAb 1F6 to the antiporter is similar to that of both the Na(+)/H(+) antiporter activity as well as to its sensitivity to trypsin. We thus suggest that these pH profiles reflect a pH-dependent conformational change, which leads to activation of the antiporter. Indeed, a replacement of Gly-338 by Ser (G338S), which alleviates the pH dependence of both the NhaA activity as well as its sensitivity to trypsin, affects in a similar pattern the binding of mAb 1F6 to NhaA. Furthermore, the binding site of mAb 1F6 is involved in the functioning of the antiporter as follows: a double Cys replacement H3C/H5C causes an acidic shift by half a pH unit in the pH dependence of the antiporter; N-ethylmaleimide, which does not inhibit the wild-type protein, inhibits H3C/H5C antiporter to an extent similar to that exerted by mAb 1F6.

A pH-dependent conformational change of NhaA Na(+)/H(+) antiporter of Escherichia coli involves loop VIII-IX, plays a role in the pH response of the protein, and is maintained by the pure protein in dodecyl maltoside.

Digestion with trypsin of purified His-tagged NhaA in a solution of dodecyl maltoside yields two fragments at alkaline pH but only one fragment at acidic pH. Determination of the amino acid sequence of the N terminus of the cleavage products show that the pH-sensitive cleavage site of NhaA, both in isolated everted membrane vesicles as well as in the pure protein in detergent, is Lys-249 in loop VIII-IX, which connects transmembrane segment VIII to IX. Interestingly, the two polypeptide products of the split antiporter remain complexed and co-purify on Ni(2+)-NTA column. Loop VIII-IX has also been found to play a role in the pH regulation of NhaA; three mutations introduced into the loop shift the pH profile of the Na(+)/H(+) antiporter activity as measured in everted membrane vesicles. An insertion mutation introducing Ile-Glu-Gly between residues Lys-249 and Arg-250 (K249-IEG-R250) and Cys replacement of either Val-254 (V254C) or Glu-241 (E241C) cause acidic shift of the pH profile of the antiporter by 0.5, 1, and 0.3 pH units, respectively. Interestingly, the double mutant E241C/V254C introduces a basic shift of more than 1 pH unit with respect to the single mutation V254C. Taken together these results imply the involvement of loop VIII-IX in the pH-induced conformational change, which leads to activation of NhaA at alkaline pH.

Purification, characterization and anticoagulant activity of a proteolytic enzyme from Vespa orientalis venom.

The anticoagulant effect of Vespa orientalis venom sac extract (VSE) was attributed to a proteolytic process, involving mainly coagulation factors VIII and IX [Joshua, H., Ishay, J., 1975. Toxicon 13, 11-20; Korenberg et al., 1988. Toxicon 26, 1169-1176]. Preliminary purification of the proteolytic activity showed the presence of three separate proteases. One of which, protease I, was purified. The purified enzyme migrated as a double band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). The molecular weights of the bands, under reduced conditions were 42 and 44 kD. Both bands retained activity after the electrophoretic run. The enzyme hydrolyses bovine factor IX (BFIX), factor X (BFX) and prothrombin. The pH optimum for the degradation of BFIX was 7.0 and its isoelectric point is above pH 10. The amino acid composition of the protease was determined.

The molecular mechanism of regulation of the NhaA Na+/H+ antiporter of Escherichia coli, a key transporter in the adaptation to Na+ and H+.

The NhaA Na+/H+ antiporter is the main system responsible for adaptation to Na+ and alkaline pH (in the presence of Na+) in Escherichia coli and many other enteric bacteria. It is under intricate control. At the protein level it is regulated directly by pH, one of its regulatory signals. A pH shift from 7 to 8.5 activates the antiporter and, in a fashion correlated with the activity change, confers a conformation change that, in isolated membrane vesicles, is reflected in the exposure of trypsin-cleavable sites. H225 and G338 are essential for the pH response of NhaA. nhaA transcription is dependent on NhaR, a positive regulator of the LysR family, and is regulated by Na+, the other environmental signal. Na+ affects the NhaR/nhaA interaction directly by changing the footprint of NhaR on nhaA in a pH-dependent fashion. The expression of nhaA is also under global regulation of H-NS. We suggest that the pattern of regulation of nhaA found in E. coli is a paradigm for the response of proteins and genes to H+ and Na+, the most common ions that challenge every cell.

A point mutation (G338S) and its suppressor mutations affect both the pH response of the NhaA-Na+/H+ antiporter as well as the growth phenotype of Escherichia coli.

pH controls the activity of the NhaA Na+/H+ antiporter of Escherichia coli. In the present work we show that replacement of glycine 338 of NhaA with serine (G338S) alleviates the pH control of the antiporter. Monitoring Na+-dependent collapse of DeltapH, to assess antiporter activity in isolated membrane vesicles, shows that the mutant protein is practically independent of pH, between pH 7 and 9, and even at pH 6 is 70% active. Similarly the purified reconstituted mutant protein catalyzes pH-independent passive efflux of 22Na from proteoliposomes as well as DeltapH-driven influx. Whereas the native NhaA in isolated membrane vesicles is exposed to digestion by trypsin only above pH 7, the mutated protein is degraded already at pH 6.5. DeltanhaA DeltanhaB cells transformed with a plasmid encoding the pH-independent antiporter are sensitive to Na+ but not to K+ at alkaline pH, while growing in the presence of both ions at neutral pH. Several possibilities that could explain the Na+ sensitivity of the mutant at alkaline pH were excluded; Western analysis and measurement of Na+/H+ antiporter activity in membrane vesicles, isolated from cells shifted to the non-permissive growth conditions, showed neither reduced expression of G338S-NhaA nor defective activity. The finding that the mutated protein is electrogenic led to the retraction of the idea that the protein is active in vitro but not in vivo at alkaline pH, when only Deltapsi exists in the cells. The Na+ concentration needed for half-maximal activity of G338S in isolated everted membrane vesicles is similar to that of the wild type. Therefore an increase in intracellular Na+ due to a reduced antiporter affinity could not explain the results. It is suggested that the loss of growth at alkaline pH in the presence of Na+ is due to the loss of the pH control of the mutated NhaA. Indeed, in the four mutations suppressing G338S phenotype, growth at alkaline pH was restored together with the pH regulation of NhaA. Three of the four suppressor mutations cluster in helix IV, whereas the original mutation is in helix XI, suggesting that the two helixes interact.

Altered Na+ and Li+ homeostasis in Saccharomyces cerevisiae cells expressing the bacterial cation antiporter NhaA.

The bacterial Na+ (Li+)/H+ antiporter NhaA has been expressed in the yeast Saccharomyces cerevisiae. NhaA was present in both the plasma membrane and internal membranes, and it conferred lithium but not sodium tolerance. In cells containing the yeast Ena1-4 (Na+, Li+) extrusion ATPase, the extra lithium tolerance conferred by NhaA was dependent on a functional vacuolar H+ ATPase and correlated with an increase of lithium in an intracellular pool which exhibited slow efflux of cations. In yeast mutants without (Na+, Li+) ATPase, lithium tolerance conferred by NhaA was not dependent on a functional vacuolar H+ ATPase and correlated with a decrease of intracellular lithium. NhaA was able to confer sodium tolerance and to decrease intracellular sodium accumulation in a double mutant devoid of both plasma membrane (Na+, Li+) ATPase and vacuolar H+ ATPase. These results indicate that the bacterial antiporter NhaA expressed in yeast is functional at both the plasma membrane and the vacuolar membrane. The phenotypes conferred by its expression depend on the functionally of plasma membrane (Na+, Li+) ATPase and vacuolar H+ ATPase.

The effect of immunodepletion of antithrombin III on the response of rabbits to Russell's viper venom-induced activation of factor X.

Many years ago it was shown that an infusion of tissue factor (TF) into rabbits causing only limited consumption of factor X and prothrombin resulted in extensive consumption of fibrinogen. More recently it was shown that an injection of a concentration of the factor X-activating fraction of Russell's viper venom (RVV-X) depleting rabbits of factor X resulted in only minimal consumption of both plasma prothrombin and fibrinogen. We report here experiments in which rabbits depleted of antithrombin III (ATIII) to different degrees were infused over 4 hours with a concentration of RVV-X, causing consumption of about 60% of plasma factor X. Similar minimal mean falls in plasma prothrombin and fibrinogen levels were observed in control rabbits given nonimmune goat IgG and in rabbits immunodepleted with goat anti-rabbit ATIII IgG to about 40% of normal plasma ATIII activity. However, if rabbits were immunodepleted to about 10% to 20% of normal plasma ATIII, then mean consumption of prothrombin was increased modestly and, more impressively, mean consumption of plasma fibrinogen was increased markedly. Whereas limited amounts of thrombin generated on the surface of phospholipid vesicles by factor VIIa/ TF can trigger extensive intravascular coagulation in rabbits with normal plasma ATIII levels, limited amounts of thrombin generated by reactions triggered by factor Xa formed in fluid phase did so only after plasma ATIII levels were markedly depleted. A possible reason for this difference is discussed.

Histidine 225, a residue of the NhaA-Na+/H+ antiporter of Escherichia coli is exposed and faces the cell exterior.

Cysteine residues were found nonessential in the mechanism of the NhaA antiporter activity of Escherichia coli. The functional C-less NhaA has provided the groundwork to study further histidine 225 of NhaA which has previously been suggested to play an important role in the activation of NhaA at alkaline pH (Rimon, A., Gerchman, Y., Olami, Y., Schuldiner, S. and Padan, E. (1995) J. Biol. Chem. 270, 26813-26817). C-less H225C was constructed and shown to possess an antiporter activity 60% of that of C-less antiporter and a pH profile similar to that of both the C-less or wild-type antiporters. Remarkably, whereas neither the wild-type nor the C-less antiporters were affected by N-ethylmaleimide, C-less H225C was inhibited by this reagent. To determine the degree of alkylation of the antiporter protein by N-ethylmaleimide, antiporter derivatives tagged at their C termini with six histidines residues were constructed. Alkylation of C-less H225C was measured by labeling of everted membrane vesicles with [14C]N-ethylmaleimide, affinity purification of the His-tagged antiporter, and determination of the radioactivity of the purified protein. This assay showed that H225C is alkylated to a much higher level than any of the native cysteinyl residues of NhaA reaching saturation at alkyl/NhaA stoichiometry of 1. The wild-type derivative showed at least 10-fold less alkylation even at higher concentrations, suggesting that H225C resides in a domain that is much more exposed to N-ethylmaleimide than the native cysteinyl residues of NhaA. Since H225C residues both in right-side out and inside-out membrane vesicles were quantitatively alkylated by N-ethylmaleimide, this assay was used to determine the accessibility of H225C to other SH reagents by titrating the H225C left free to react with N-ethylmaleimide, following exposure of the membranes to the reagents. Furthermore, since membrane-impermeant probes can react with residues in membrane-embedded protein only if accessible to the medium containing the reagent, the assay was used to determine the membrane topology of H225C. As expected for a membrane-permeant probe, p-chloromercuribenzoate reacted with H225C as efficiently as N-ethylmaleimide in both membrane orientations. Similar results were obtained with methanethiosulfonate ethylammonium supporting the recent observations that this probe is membrane-permeant. On the other hand, both membrane-impermeant reagents p-chloromercuribenzosulfonate and methanethiosulfonate ethyl-trimethyl ammonium bromide reacted with H225C 10-fold more in right-side out than in inside-out vesicles, and p-chloromercuribenzosulfonate also blocked completely the H225C in intact cells. These results strongly suggest that H225C is exposed at the periplasmic face of the membrane.

Na+-induced transcription of nhaA, which encodes an Na+/H+ antiporter in Escherichia coli, is positively regulated by nhaR and affected by hns.

nhaA encodes an Na+/H+ antiporter in Escherichia coli which is essential for adaptation to high salinity and alkaline pH in the presence of Na+. We used Northern (RNA) analysis to measure directly the cellular levels of nhaA mRNA. NhaR belongs to the LysR family of regulatory proteins. Consistent with our previous data with an nhaA'-'lacZ fusion, NhaR was found to be a positive regulator and Na+ was found to be a specific inducer of nhaA transcription. In the nhaA'-'lacZ fusion, maximal induction was observed at alkaline pH. In contrast, in the nhaA+ strain both the level of nhaA expression and the induction ratio were lower at alkaline pH. This difference may be due to the activity of NhaA in the wild-type strain as NhaA efficiently excreted Na+ at alkaline pH and reduced the intracellular concentration of Na+, the signal for induction. We also showed that although the global regulator rpoS was not involved in nhaA regulation, the global regulator hns played a role. Thus, the expression of nhaA'-'lacZ was derepressed in strains bearing hns mutations and transformation with a low-copy-number plasmid carrying hns repressed expression and restored Na+ induction. The derepression in hns strains was nhaR independent. Most interestingly, multicopy nhaR, which in an hns+ background acted only as an Na+-dependent positive regulator, acted as a repressor in an hns strain in the absence of Na+ but was activated in the presence of the ion. Hence, an interplay between nhaR and hns in the regulation of nhaA was suggested.

Replacements of histidine 226 of NhaA-Na+/H+ antiporter of Escherichia coli. Cysteine (H226C) or serine (H226S) retain both normal activity and pH sensitivity, aspartate (H226D) shifts the pH profile toward basic pH, and alanine (H226A) inactivates the carrier at all pH values.

We have previously shown that replacement of His-226 in the NhaA Na+/H+ antiporter of Escherichia coli to Arg (H226R) shifts the pH profile of the antiporter toward acidic pH and as a result of delta nhaA delta nhaB strain bearing this mutation is Na+ sensitive at alkaline pH (Gerchman, Y., Olami, Y., Rimon, A., Taglicht, D., Schuldiner, S. and Padan, E. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1212-1216). In the present work the role of His-226 in the response of NhaA to pH has been studied in detail. The Na+ sensitivity of the delta nhaA delta nhaB mutant bearing the H226R-NhaA plasmid at alkaline pH provided a very powerful tool to isolate revertants and suppressants of H226R growing on high Na+ at alkaline pH. With this approach cysteine (H226C) and serine (H226S) replacements were found to efficiently replace His-226 and yield an antiporter, which like the wild-type protein, is activated by pH between pH 7 and 8. These results imply that polarity and/or hydrogen bonding, the common properties shared by these amino acid residues, are essential at position 226 for pH regulation of NhaA. This suggestion was substantiated by site-directed mutagenesis of His-226 either to alanine (H226A) or aspartate (H226D). Whereas H226A-NhaA shows very low activity which is not activated by pH, H226D-NhaA is active and regulated by pH. The pH profile of H226D is shifted by half a pH unit toward alkaline pH, as opposed to the previously isolated mutant H226R which has a pH profile shift, to the same extent, but toward acidic pH. It is suggested that charge modifies the pH profile but is not essential for the pH regulation of NhaA.

Quantitative ultrasound of the tibia: a novel approach for assessment of bone status.

An ultrasound instrument has recently been developed for the diagnosis and monitoring of osteoporosis (SoundScan 2000, Myriad Ultrasound Systems Ltd., Israel). The instrument measures the speed of propagation of ultrasound waves (SOS, meters per second) along a fixed longitudinal distance of the cortical layer at the tibial shaft. Its in vivo precision is 0.25%. The performance of the SoundScan 2000 was studied in 307 Caucasian women (age range 24-87 years) who also had their bone mineral density (BMD) measured at the spine, femoral neck, and radial shaft by absorptiometric techniques. The SOS ranged from 3471-4226 m/sec (mean 3867). The standardized coefficient of variation (CV), an expression of the effective clinical precision corrected for the spread of measurements (CV/[range/mean]), was 1.6% for the tibial SOS, compared to 1.5%, 3.8%, and 4.4% for spinal, femoral, and radial BMD, respectively. Tibial SOS significantly correlated with age (r = -0.52), time since menopause (r = -0.43), height (r = 0.29), and weight (r = 0.16), as well as with BMD at the radius (r = 0.63), spine (r = 0.50), and femur (r = 0.47). After classification of bone measurements into tertiles, about 60% of the women with low tertile spinal BMD fell within the low tertile of either tibial SOS, femoral BMD, or radial BMD. The results show that measurement of tibial SOS is a precise method of assessing bone status without exposing the patient to sources of radiation.

Purification properties and specificity of cathepsin D from Cyprinus carpio.

Cathepsin D was purified 750-fold from a homogenate of Cyprinus carpio muscles. The purified enzyme has a molecular weight of 36,000, is inhibited by pepstatin and is active between pH 2.7 and 3.7 when tested on hemoglobin as the substrate. It consists of two isoenzymes with pIs of 5.65 and 6.1, respectively. The mode of cleavage of the beta chain of oxidized insulin was determined by analysis of the N-terminal amino acids of the cleaved peptides. The major points of cleavage of the beta chain of oxidized insulin are 56% at Tyr16-Leu17 and 40% at Phe25-Tyr26. The minor points of cleavage are at Leu15-Tyr16, Phe24-Phe25, Gly23-Phe24, Leu11-Val12, Ala14-Leu15 and Gln4-His5.

Histidine-226 is part of the pH sensor of NhaA, a Na+/H+ antiporter in Escherichia coli.

The nhaA gene of Escherichia coli, which encodes a pH-activated Na+/H+ antiporter, has been modified; six of its eight histidine codons were mutated to arginine codons by site-directed mutagenesis, yielding the mutations H254R-H257R (a double mutant), H226R, H39R, H244R, and H319R. In addition a deletion (delta nhaA1-14) lacking the remaining two histidines, His-3 and His-5, has been constructed. By comparing the phenotypes conferred by plasmids bearing the various mutations to the phenotype of the wild type upon transformation of strains NM81 (delta nhaA) or EP432 (delta nhaA, delta nhaB) we found that none of the NhaA histidines are essential for the Na+/H+ antiporter activity of the NhaA protein. However, the replacement of His-226 by Arg markedly changes the pH dependence of the antiporter. All mutants except H226R confer to NM81 and EP432 Na+ resistance up to pH 8.5 as well as Li+ resistance. Cells bearing H226R are resistant to Li+ and to Na+ at neutral pH, but they become sensitive to Na+ above pH 7.5. Analysis of the Na+/H+ antiporter activity of membrane vesicles derived from H226R cells shows that the mutated protein is activated by pH to the same extent as the wild type. However, whereas the activation of the wild-type NhaA occurs between pH 7 and pH 8, that of H226R antiporter occurs between pH 6.5 and pH 7.5. Furthermore, while the wild-type antiporter remains almost fully active at least up to pH 8.5, H226R is reversibly inactivated above pH 7.5, reaching 10-20% of the maximal activity at pH 8.5. We suggest that His-226 is part of a pH-sensitive site that regulates the activity of NhaA.

Activation of the coagulation mechanism on tumor necrosis factor-stimulated cultured endothelial cells and their extracellular matrix. The role of flow and factor IX/IXa.

Infusion of tumor necrosis factor (TNF) into tumor-bearing mice led to intravascular clot formation with fibrin deposition in microvessels in the tumor bed in close association with the vessel wall, which could be prevented by active site-blocked factor IXa (IXai). This observation prompted us to examine the role of the intrinsic system in activation of the coagulation mechanism on TNF-stimulated human endothelial cell monolayers and endothelial-derived matrix during exposure to purified coagulation factors or flowing blood. Treatment of endothelial cells in intact monolayers with TNF induced expression of the procoagulant cofactor tissue factor (TF) in a dose-dependent manner, and after removal of the cells, TF was present in the matrix. TNF-treated endothelial cell monolayers exposed to blood anticoagulated with low molecular weight heparin induced activation of coagulation. Addition of IXai blocked the procoagulant response on TNF-treated endothelial cells, and consistent with this, the presence of factor IX/VIIIa enhanced endothelial TF/factor VII(a) factor X activation over a wide range of cytokine concentrations (0-600 pM). When TF-dependent factor X activation on endothelial cells was compared with preparations of subendothelium, the extracellular matrix was 10-20 times more effective. IXai blocked TF/factor VII(a) mediated activated coagulation on matrix, but only at lower concentration of TNF (less than 50 pM). Similarly, enhancement of factor Xa formation on matrix by factors IX/VIIIa was most evident at lower TNF concentrations. When anticoagulated whole blood flowing with a shear of 300 s-1 was exposed to matrices from TNF-treated endothelial cells, but not matrices from control cells, fibrinopeptide A (FPA) generation, fibrin deposition, and platelet aggregate formation were observed. FPA generation could be prevented by a blocking antibody to TF and by active site-blocked factor Xa (Xai) over a wide range of TNF concentrations (0-600 pM), whereas IXai only blocked FPA generation at lower TNF concentrations (less than 50 pM). Activation of coagulation on matrix from TNF-stimulated endothelial cells was dependent on the presence of platelets, indicating the important role of platelets in propagating the reactions leading to fibrin formation. These observations demonstrate the potential of cytokine-stimulated endothelium and their matrix to activate coagulation and suggest the importance of the intrinsic system in factor Xa formation on cellular surfaces.