Nucleotide receptor P2RX7 stimulation enhances LPS-induced interferon-ß production in murine macrophages.

Stimulation of P2RX(7) with extracellular ATP potentiates numerous LPS-induced proinflammatory events, including cytokine induction in macrophages, but the molecular mechanisms underlying this process are not well defined. Although P2RX(7) ligation has been proposed to activate several transcription factors, many of the LPS-induced mediators affected by P2RX(7) activation are not induced by P2RX(7) agonists alone, suggesting a complementary role for P2RX(7) in transcriptional regulation. Type I IFN production, whose expression is tightly controlled by multiple transcription factors that form an enhanceosome, is critical for resistance against LPS-containing bacteria. The effect of purinergic receptor signaling on LPS-dependent type I IFN is unknown and would be of great relevance to a diverse array of inflammatory conditions. The present study demonstrates that stimulation of macrophages with P2RX(7) agonists substantially enhances LPS-induced IFN-ß expression, and this enhancement is ablated in macrophages that do not express functional P2RX(7) or when the MAPK MEK1/2 pathways are inhibited. Potentiation of LPS-induced IFN-ß expression following P2RX(7) stimulation is likely transcriptionally regulated, as this enhancement is observed at the IFN-ß promoter level. Furthermore, P2RX(7) stimulation is able to increase the phosphorylation and subsequent IFN-ß promoter occupancy of IRF-3, a transcription factor that is critical for IFN-ß transcription by TLR agonists. This newly discovered role for P2RX(7) in IFN regulation may have implications in antimicrobial defense, which has been linked to P2RX(7) activation in other studies.

The effect of mindfulness meditation on cough reflex sensitivity.

BACKGROUND: Chronic cough is common, and medical treatment can be ineffective. Mindfulness is a psychological intervention that aims to teach moment-to-moment non-judgemental awareness of thoughts, feelings and sensations. METHOD: 30 healthy subjects and 30 patients with chronic cough were studied in two sequential trials. For both studies, cough reflex sensitivity to citric acid (C5) was measured on two occasions, with urge to cough rated following each inhalation; between challenges subjects were randomised to (1) no intervention, (2) mindfulness or (3) no intervention but modified cough challenge (subjects suppress coughing). For the healthy volunteers, measures were 1 h apart and mindfulness was practised for 15 min. For the patients with chronic cough measures were 1 week apart and mindfulness was practised daily for 30 min. RESULTS: In healthy volunteers, median change (interquartile range (IQR)) in cough reflex sensitivity (logC5) for no intervention, mindfulness and suppression was +1.0 (0.0 to +1.3), +2.0 (+1.0 to +3.0) and +3.0 (+2.8 to +3.0) doubling concentrations (p = 0.003); there were significant reductions for both mindfulness (p = 0.043) and suppression (p = 0.002) over no intervention. In patients with cough, median change (IQR) in logC5 for no intervention, mindfulness training and voluntary suppression was 0.0 (-1.0 to +1.0), +1.0 (-0.3 to +1.0) and +1.0 (+1.0 to +2.0) doubling concentrations (p = 0.046); there was a significant reduction for suppression (p = 0.02) but not mindfulness (p = 0.35). Urge to cough did not change after mindfulness compared with control in either healthy subjects (p = 0.33) or those with chronic cough (p = 0.47). CONCLUSION: Compared with control, mindfulness decreased cough reflex sensitivity in healthy volunteers, but did not alter cough threshold in patients with chronic cough. Both groups were able to suppress cough responses to citric acid inhalation.

Surgical practice in the UK.

Throughout the working lifetime of any surgeon, major changes take place often stimulated by matters outwith the practitioner's control. This has certainly been true over the last forty years. The holistic care of a patient is now well established, with surgeons being seen as doctors who can add technical skills to their therapeutic armamentarium rather than being speedy technicians. Many of the changes have been for the good of patient care and developments in other areas such as anaesthesia, intensive care and radiology have had a significant influence on surgical practice. Not all change has been for the better. Some of the Government pressures and concerns with financing of the Health Service have had an adverse affect by deviating attention away from the patient.

The pollination ecology of Grevillea beadleana McGillivray, an endangered shrub from northern New South Wales, Australia.

Grevillea beadleana (Proteaceae) is an endangered species known from five populations in northern New South Wales, Australia. The reproductive ecology of G. beadleana was compared in two populations with a ten-fold difference in the number of plants. Grevillea beadleana was found to be self-compatible in both populations and an examination of pollen viability and stigma maturation revealed that the species is protandrous. Flowering within inflorescences is acropetallous. In the first season plants in the largest population produced approx. ten-fold more inflorescences than those in the smaller population and, although the number of flowers per inflorescence did not vary significantly between populations the first season, the larger population produced more fruit per inflorescence than the smaller population. However, fruit to flower ratios were less than 0.2 in both seasons and populations. In both populations the number of fruit was significantly greater at the proximal end of the inflorescence, where flowers open first, compared with medial and distal positions. Several bird species were observed visiting flowers, although few birds were recorded foraging at plants in the smaller population. Within both populations, birds tended to make more within- than between-plant visits. Self-compatibility, acropetally and proximal fruit-set, combined with the predominantly within-plant movement of honeyeaters, suggests inbreeding may be common within both populations of G. beadleana. Pollination and fruiting success are discussed for G. beadleana and breeding systems among rare and common taxa in Grevillea are reviewed.

Characterization of specific IgE response in vitro against protein and drug allergens using atopic and normal donors.

BACKGROUND: As the incidence of allergy to different compounds increases in society, the need to understand and characterize specific IgE responses becomes obvious. Different cell culture systems have been evaluated for their ability to support such IgE secretion. METHODS: One system employed human peripheral lymphocytes (PBL) from normal donors stimulated with anti-CD3 activated T cells with or without the presence of allergens like benzylpenicillin (BP) and Phlenum pratense (PP). Secretion of IgE was analyzed in ELISA and compared to the IgG response to the nonallergenic antigen tetanus toxoid (TT). Another system employed stimulation of T and B cells with a heterotope, consisting of a T helper cell epitope derived from TT, and a B cell allergen epitope derived from BP. The specific IgE secretion was compared, using lymphocytes from normal as well as BP-allergic donors. RESULTS: Anti-CD3 stimulated T cells supported BP-specific IgE secretion in six of 11 normal donors. This response was inhibited in four donors and enhanced in two donors by the addition of the BP-allergen to the culture. In contrast, addition of the protein allergen (PP) or antigen (TT) to the same culture system inhibited both IgE and IgG synthesis in all experiments. Cells from the majority (10/16) of the BP-allergic donors failed to produce BP-specific IgE in vitro, when cultured in the presence of allergen. CONCLUSIONS: An allergen specific immune response is readily generated in vitro. The differential response against benzylpenicillin between different donor categories most probably reflects the level of pre-exposure to this allergen in vivo.

Intraoperative nerve stimulation predicts postoperative potency.

OBJECTIVES: Intraoperative nerve stimulation has been used to map the course of cavernosal nerve bundles to aid in nerve-sparing radical prostatectomy (RP). We sought to determine whether the intraoperative nerve stimulation response after removal of the prostate specimen predicts postoperative potency. METHODS: We prospectively evaluated 63 consecutive patients undergoing RP. Among these 63 patients, 41 patients had a bilateral nerve-sparing procedure, and 22 underwent bilateral wide resection of the neurovascular bundles. Potency was determined at 1 year by direct patient interview. RESULTS: Of the 22 patients with wide resection, 16 (73%) had no observed stimulated response, 4 (18%) had a measurable tumescence response, and 2 (9%) had detumescence. All 22 patients were impotent postoperatively. At a minimum follow-up of 1 year, of the 41 patients who underwent nerve-sparing RP, 27 (66%) had postoperative erections sufficient for penetration. Of the 30 men with an intraoperative stimulated tumescence response, 24 (80%) were potent; of the 6 with a detumescence response, 2 (33%) were potent; and of the 5 with no stimulated nerve response, 1 (20%) was potent. A tumescence response was significantly more predictive of postoperative potency than no intraoperative response (P = 0.017). CONCLUSIONS: An intraoperative tumescence response with stimulation of the neurovascular bundles after prostate specimen removal is more likely to correspond to successful postoperative sexual function after nerve-sparing RP than is no response.

Inhibition of export of fibroblast growth factor-2 (FGF-2) from the prostate cancer cell lines PC3 and DU145 by Anvirzel and its cardiac glycoside component, oleandrin.

Anvirzel is an extract of Nerium oleander currently undergoing Phase I clinical evaluation as a potential treatment for cancer. Two of the active components of Anvirzel are the cardiac glycosides oleandrin and oleandrigenin. Previous studies have demonstrated that, in vitro, cardiac glycosides may inhibit fibroblast growth factor-2 (FGF-2) export through membrane interaction with the Na(+),K(+)-ATPase pump. In continuing research on the antitumor activity of this novel plant extract, the relative abilities of oleandrin and oleandrigenin to inhibit FGF-2 export from two human prostate cancer cell lines, DU145 and PC3, were examined. An ELISA assay was utilized to determine the FGF-2 concentration in the cell culture medium before and after exposure to cardiac glycosides or the parent extract material Anvirzel. Both cell lines were exposed to non-cytotoxic concentrations of oleandrin (0.05 and 0.1 ng/mL) for up to 72 hr. Studies also were conducted with Anvirzel and ouabain. Oleandrin (0.1 ng/mL) produced a 45.7% inhibition of FGF-2 release from PC3 cells and a 49.9% inhibition from DU145 cells. Non-cytotoxic concentrations (100 ng/mL) of Anvirzel produced a 51.9 and 30.8% inhibition of FGF-2 release, respectively, in the two cell lines. The decrease in FGF-2 release from cells required continuous incubation for 48--72 hr; shorter incubation times were not effective. These results demonstrate that Anvirzel, like oleandrin, inhibited FGF-2 export in vitro from PC3 and DU145 prostate cancer cells in a concentration- and time-dependent fashion and may, therefore, contribute to the antitumor activity of this novel treatment for cancer.

Post-infective diarrhoea.

When diarrhoea caused by gastroenteritis persists for more than two weeks it is referred to as persistent diarrhoea in developing countries. Whilst the Control of Diarrhoeal Diseases programme has decreased mortality from acute diarrhoea, mortality from persistent diarrhoea has not been so responsive. A number of factors have been identified which are determinants for the progression of an acute episode to one which persists in developing communities. In one study from west Africa, current infection with Cryptosporidium parvum was the most significant factor. In studies from Brazil and India, continuing infection with enteropathogenic Escherichia coli was identified in 50% of infants with persistent diarrhoea. Persistent small intestinal mucosal damage is of key importance in such children. Management of established cases is complex and difficult. However, there is clear evidence that zinc is involved in the recovery of small intestinal mucosa after injury. Zinc supplementation may indeed significantly reduce the duration of persistent diarrhoea. However, the whole question of public health supplementation with zinc, vitamin A, or other supplements, is contentious at present.

A pilot study of N-acetyl glucosamine, a nutritional substrate for glycosaminoglycan synthesis, in paediatric chronic inflammatory bowel disease.

BACKGROUND: The breakdown of glycosaminoglycans is an important consequence of inflammation at mucosal surfaces, and inhibition of metalloprotease activity may be effective in treating chronic inflammation. AIM: To report an alternative approach, using the nutriceutical agent N-acetyl glucosamine (GlcNAc), an amino-sugar directly incorporated into glycosaminoglycans and glycoproteins, as a substrate for tissue repair mechanisms. METHODS: GlcNAc (total daily dose 3-6 g) was administered orally as adjunct therapy to 12 children with severe treatment-resistant inflammatory bowel disease (10 Crohn's disease, 2 ulcerative colitis). Seven of these children suffered from symptomatic strictures. In addition, similar doses were administered rectally as sole therapy in nine children with distal ulcerative colitis or proctitis resistant to steroids and antibiotics. Where pre- and post-treatment biopsies were available (nine cases), histochemical assessment of epithelial and matrix glycosaminoglycans and GlcNAc residues was made. FINDINGS: Eight of the children given oral GlcNAc showed clear improvement, while four required resection. Of the children with symptomatic Crohn's stricture, only 3 of 7 have required surgery over a mean follow-up of > 2.5 years, and endoscopic or radiological improvement was detected in the others. Rectal administration induced remission in two cases, clear improvement in three and no effect in two. In all cases biopsied there was evidence of histological improvement, and a significant increase in epithelial and lamina propria glycosaminoglycans and intracellular GlcNAc. CONCLUSIONS: GlcNAc shows promise as an inexpensive and nontoxic treatment in chronic inflammatory bowel disease, with a mode of action which is distinct from conventional treatments. It may have the potential to be helpful in stricturing disease. However, controlled trials and an assessment of enteric-release preparations are required to confirm its efficacy and establish indications for use.

Short-term risedronate treatment in postmenopausal women: effects on biochemical markers of bone turnover.

The development of new biochemical markers has made it possible to assess the effects of therapeutic agents on bone turnover more rapidly and precisely. In this early phase II study, we analyzed the effects of short-term, high-dose treatment with risedronate, a potent pyridinyl bisphosphonate, on markers of bone resorption and formation. Resorption markers included urinary free deoxypyridinoline (D-Pyr) crosslinks, N-terminal telopeptide (NTx) and C-terminal telopeptide (CTx) type I collagen crosslinks. Bone formation markers included osteocalcin (OC), bone-specific alkaline phosphatase (BSAP) and the C-terminal peptide of type I procollagen (PICP). All three resorption markers showed rapid, significant (p<0.05) decreases from baseline following daily administration of 30 mg risedronate for 2 weeks. The mean decreases at 2 weeks were 28% for D-Pyr, 61% for NTx and 73% for CTx, respectively. Over the next 10 weeks after treatment, D-Pyr approached baseline while NTx and CTx remained well below baseline values. The markers of bone formation showed little change during therapy but decreased significantly at 4-10 weeks after therapy - an expected outcome of bisphosphonate therapy. Moreover, there was a significant correlation between the early effects on bone resorption markers and the delayed effects on formation markers. This study demonstrates that the approved dose of risedonate (30 mg/day) for Paget's disease is effective at decreasing bone turnover after 2 weeks of treatment, as observed by the sensitive response of bone turnover markers.

Early post-prostatectomy pelvic floor biofeedback.

PURPOSE: We determined whether biofeedback enhanced pelvic floor exercises begun 6 weeks after radical prostatectomy improve the early recovery of continence. MATERIALS AND METHODS: We randomized 30 patients who underwent radical retropubic prostatectomy into a group that received 5 biofeedback sessions and a control group. RESULTS: Overall 87% of patients were pad-free at 6 months with similar results in the treatment and control groups (86% versus 88%). There was no statistically significant difference in pad test results or voiding diary records in the 2 groups. CONCLUSIONS: A treatment program of biofeedback enhanced pelvic floor exercises begun 6 weeks after radical retropubic prostatectomy did not significantly affect continence in this study.

Does MK-801 discrimination constitute an animal model of schizophrenia useful for detecting atypical antipsychotics?

Two groups of female Wistar rats were trained to discriminate two doses (0.075 and 0.0375 mg/kg) of the noncompetitive NMDA antagonist MK-801 (dizocilpine) in a food-rewarded operant FR30 drug discrimination task. The atypical neuroleptic clozapine (2-6 mg/kg) produced only minimal antagonism (max. 32%) of the MK-801 cue at either training dose, and the "antagonist" effects were not clearly dose related. Furthermore, in the 0.075 mg/kg trained animals clozapine at 3 mg/kg failed to shift the MK-801 dose-response curve to the right. The alpha1-adrenoceptor antagonist prazosin (1-8 mg/kg) was also tested for antagonism of the 0.0375 mg/kg MK-801 cue, and again, only partial antagonism was seen (maximum 36%). Recently, it was suggested [4] that as the discriminative stimulus produced by MK-801 (0.075 mg/kg) was fully antagonized by clozapine at 3 mg/kg, but not by the typical neuroleptic haloperidol, this assay may be a useful screen for detecting atypical neuroleptics. It would seem, however, that this is not necessarily the case, and that the MK-801 discriminative cue may not be psychotomimetic. However, as this was a food rewarded rather than an avoidance paradigm that was used in the prior study [4], it may be that the drug discrimination procedure itself is a critical factor, although this hypothesis requires empirical testing.

The effects of triamcinolone acetonide aqueous nasal spray on adrenocortical function in children with allergic rhinitis.

BACKGROUND: Suppression of adrenocortical function, a risk associated with oral corticosteroids, is minimized with intranasal corticosteroids. Triamcinolone acetonide (TAA) aqueous nasal spray, at therapeutic doses, has no measurable effect on adrenocortical function in adults with allergic rhinitis. OBJECTIVE: This double-blind, placebo-controlled study compared the effect of once-daily TAA aqueous nasal spray (220 or 440 microg) with placebo on adrenocortical function after 6 weeks of treatment in pediatric (children 6 to 12 years of age) patients with allergic rhinitis. The pharmacokinetic profile of TAA was examined after once-daily intranasal administration of TAA aqueous nasal spray 440 microg for 6 weeks. METHODS: Eighty children received TAA aqueous nasal spray 220 microg or 440 microg or placebo for 6 weeks. Adrenocortical function was assessed by analyzing plasma cortisol levels before stimulation (0 hour) and at 30 and 60 minutes after a rapid 1-hour intravenous cosyntropin stimulation test performed before treatment and after 6 weeks of treatment. Samples for pharmacokinetic evaluation were collected from 19 patients at baseline (0 hour) and at 0.5, 1, 1.5, and 6 hours after the final dose of study medication. RESULTS: After 6 weeks, no significant effects on adrenocortical function were observed at 30 or 60 minutes after cosyntropin stimulation with either dose of TAA aqueous nasal spray. TAA concentrations in plasma showed rapid elimination of the drug, with little or no accumulation. CONCLUSIONS: TAA aqueous nasal spray (220 or 440 microg/day) has no measurable effect on adrenocortical function in pediatric patients with allergic rhinitis. Pharmacokinetic parameters after 440 microg/day of TAA aqueous nasal spray indicate a rapid decline of plasma drug levels, with little or no systemic accumulation of study drug.

Nutrition in inflammatory bowel disease.

Nutrition is clearly disturbed by active intestinal inflammation. Appetite is reduced, yet energy substrates are diverted into the inflammatory process, and thus weight loss is characteristic. The nutritional disturbance represents part of a profound defect of somatic function. Linear growth and pubertal development in children are notably retarded, body composition is altered, and there may be significant psychosocial disturbance. Macrophage products such as tumour necrosis factor-alpha and interleukins-1 and 6 may be the central molecules that link the inflammatory process to this derangement of homeostasis. Intriguingly, there is also increasing evidence that an aggressive nutritional programme may in itself be sufficient to reduce the mucosal inflammatory response. Recent evidence suggests that enteral nutrition alone may reduce many pro-inflammatory cytokines to normal and allow mucosal healing. In addition, specific nutritional components, such as n-3 polyunsaturated fatty acids, may have an anti-inflammatory effect as they may alter the pattern of leukotrienes generated during the immune response. The recent discovery of the specific molecular mediators of appetite and body composition, such as leptin and myostatin, may allow increased therapeutic specificity and further improvement in the nutritional treatment of the inflammatory bowel diseases.

Blood loss during radical retropubic prostatectomy: is preoperative autologous blood donation indicated?

PURPOSE: We assessed the requirement for transfusion of allogeneic blood in a contemporary series of patients who did not deposit autologous blood before radical retropubic prostatectomy. MATERIALS AND METHODS: After a policy was adopted in which preoperative autologous blood was not donated, 124 consecutive patients underwent radical retropubic prostatectomy. Type and screen for allogeneic blood were routinely available but neither hemodilation nor a cell saver was used. RESULTS: Mean intraoperative blood loss was 579 cc and mean postoperative serum hematocrit was 33%. Only 3 patients (2.4%) required blood products due to intraoperative blood loss (2) and postoperative bleeding from a duodenal ulcer (1). CONCLUSIONS: Transfusion of blood products was required in a small percentage of our patients even without autologous blood donation. Therefore, the overall cost of care is decreased but, more importantly, the potential risks associated with autologous or allogeneic blood transfusion are eliminated.

Developmental expression of alpha 7 neuronal nicotinic receptor messenger RNA in rat sensory cortex and thalamus.

The distribution of alpha 7 messenger RNA expression was characterized in developing rat cortex and thalamus. Northern blot analysis of neonatal and adult cortex revealed a single messenger RNA transcript of 5.7 kb. Using in situ hybridization with both full length and short 35S-labeled alpha 7 riboprobes, a distinct transient expression of messenger RNA within sensory cortex and thalamus, during early postnatal development, was observed. alpha 7 transcripts were expressed in low levels as early as embryonic day 13 in the ventricular zone of the neocortex, and as early as embryonic day 15 in the thalamic neuroepithelium. A marked increase in messenger RNA levels was observed during the late prenatal period in both sensory and non-sensory regions of the cortex and thalamus. Moderate to high levels of messenger RNA were maintained into the first postnatal week, followed by a decline into adulthood. alpha 7 messenger RNA expression was significantly higher in the anterodorsal, lateral dorsal, ventral posterior medial and ventral posterior lateral thalamic nuclei of postnatal day 7 pups than in adult brains. Expression of messenger RNA within dorsal lateral geniculate, ventral lateral geniculate and medial geniculate did not show a significant reduction with age. Within the developing cortex, messenger RNA expression delineated the primary somatosensory, auditory and visual cortices in a unique laminar pattern that was consistently and significantly higher than in the adult in superficial layer VI. Higher levels of expression were also observed in retrosplenial cortex at postnatal day 7 than in the adult. Tangential sections through postnatal day 7 cortex revealed low levels of alpha 7 messenger RNA expression delineating the primary sensory areas in layer IV, corresponding to acetylcholinesterase-labeled thalamocortical afferents. However, these sensory areas exhibited higher levels of alpha 7 messenger RNA expression and were more clearly defined in layer VI, but not by acetylcholinesterase staining. The distribution of alpha 7 messenger RNA within the developing thalamocortical system parallels the distribution of alpha-bungarotoxin binding sites and suggests that the receptor is localized on both thalamic cells and their cortical target neurons. This transient and distinct pattern of distribution of the alpha 7 neuronal nicotinic receptor, which coincides with the major phase of thalamocortical development, suggests that it may play a functional role in the development of cortical circuitry.

Polymeric nutrition as the primary therapy in children with small bowel Crohn's disease.

BACKGROUND: Recent studies in adults have shown that polymeric (whole protein) diets are as effective as semi-elemental and elemental formulae for the induction of remission in small bowel Crohn's disease. Whole protein diets are more palatable and cheaper. There have been no studies confirming efficacy in children. PATIENTS AND METHODS: We report our experience with seven children with active small bowel Crohn's disease given a casein-based, polymeric feed rich in TGF-beta 2 (Specific Polymeric Diet; Nestle-Clintec; Vevey, Switzerland) as complete nutrition for 8 weeks. RESULTS: Initial and follow-up assessments were performed. All children showed a significant improvement in disease activity, with C-reactive protein returning to normal, an increase in serum albumin and a good weight gain. Initial and follow-up ileal biopsies were assessed and showed reduced mucosal inflammation in six of seven children, with complete healing in two. CONCLUSION: In an uncontrolled descriptive study we have shown that a polymeric (whole protein) diet is a therapeutic option for small bowel Crohn's disease in children. By comprehensive follow-up we have demonstrated clinical and biochemical remission, with an improved endoscopic appearance and a reduction of mucosal inflammation in the terminal ileum.

Dicarboxylate transport at the vacuolar membrane of the CAM plant Kalanchoë daigremontiana: sensitivity to protein-modifying and sulphydryl reagents.

Malate is widespread as a charge-balancing anion in plant vacuoles and plays a central role in nocturnal CO2 assimilation in crassulacean acid metabolism (CAM). To characterize the malate transport system at the vacuolar membrane of CAM plants, tonoplast vesicles were prepared from leaf mesophyll cells of the crassulacean plant Kalanchoë daigremontiana. Dicarboxylate uptake, assayed by a membrane-filtration method using [14C]malate or [14C]succinate, displayed saturation kinetics with apparent Km values of 4.0 mM (malate) and 1.8 mM (succinate); competition experiments indicated that both anions were transported by the same system. Dicarboxylate uptake was stimulated severalfold by activation of the tonoplast H(+)-ATPase or H(+)-PPiase, an effect inhibitable by ionophore. Passive (non-energized) dicarboxylate uptake was sensitive to the sulphydryl reagents N-ethylmaleimide and p-chloromercuribenzene sulphonate, as well as to a range of protein modifiers. In particular, inhibition by pyridoxal phosphate was completely substrate-protectable, and that by phenylglyoxal partially so, thus implicating at least one lysine residue and perhaps also an arginine residue in the substrate-recognition site of the transport protein. The involvement of one or more critical lysine residue was supported by analysis of the initial phase of inhibition by pyridoxal phosphate: this showed pseudo-first-order kinetics with a reaction order of 1.03 +/- 0.13 and a Kd for substrate protection close to the apparent Km for dicarboxylate uptake.