Enhanced Carbapenemase Producing Enterobacterales (CPE) Screening in a Paediatric Population.

Aims The aim of this period of extended screening (to include those being readmitted to TSCUH if they had been an inpatient in the preceding year) was to determine whether any additional CPE positive patients were identified. Methods Education was given to Clinical Nurse Managers regarding those requiring screening and reinforced at daily handover meetings. Results 917 patients were screened during the four-month period; only two positive patients were identified, who would have been screened under the previous local guidelines. Conclusion The screening of an additional 314 patients (34% increase compared with the same period in the previous year) yielded no positive results, showing that patients whose only risk factor was admission to TSCUH within the previous year do not currently require screening.

Agreement and consistency in the triaging of musculoskeletal primary care referrals by vetting clinicians using a knowledge-based triage tool.

BACKGROUND: Primary care referrals received by secondary care services are vetted or triaged to pathways best suited for patients' needs. If knowledge-based triaging is used by vetting clinicians, accuracy is required to avoid incorrect decisions being made. With limited evidence to support best practice, we aimed to evaluate consistency across vetting clinicians' decisions and their agreement with a criterion decision. METHODS: Twenty-nine trained vetting clinicians (18 female) representative of pay grades independently triaged five musculoskeletal physiotherapy referral cases into one of 10 decisions using an internally developed triage tool. Agreement across clinicians' decisions between and within cases was assessed using Fleiss's kappa overall and within pay grade. Proportions of triage decisions consistent with criterion decisions were assessed using Cochran's Q test. RESULTS: Clinician agreement was fair for all cases (κ = 0.385) irrespective of pay grade but varied within clinical cases (κ = -0.014-0.786). Proportions of correct triage decisions were significantly different across cases [Q(4) = 33.80, P < 0.001] ranging from 17% to 83%. CONCLUSIONS: Agreement and consistency in decisions were variable using the tool. Ensuring referrer information is accurate is vital, as is developing, automating and auditing standards for certain referrals with clear pathways. But we argue that variable vetting outcomes might represent healthy pathway abundance and should not simply be automated in response to perceived inefficiencies.

Diversity of channels generated by different combinations of epithelial sodium channel subunits.

The epithelial sodium channel is a multimeric protein formed by three homologous subunits: alpha, beta, and gamma; each subunit contains only two transmembrane domains. The level of expression of each of the subunits is markedly different in various Na+ absorbing epithelia raising the possibility that channels with different subunit composition can function in vivo. We have examined the functional properties of channels formed by the association of alpha with beta and of alpha with gamma in the Xenopus oocyte expression system using two-microelectrode voltage clamp and patch-clamp techniques. We found that alpha beta channels differ from alpha gamma channels in the following functional properties: (a) alpha beta channels expressed larger Na+ than Li+ currents (INa+/ILi+ 1.2) whereas alpha gamma channels expressed smaller Na+ than Li+ currents (INa+/ILi+ 0.55); (b) the Michaelis Menten constants (Km of activation of current by increasing concentrations of external Na+ and Li+ of alpha beta channels were larger (Km > 180 mM) than those of alpha gamma channels (Km of 35 and 50 mM, respectively); (c) single channel conductances of alpha beta channels (5.1 pS for Na+ and 4.2 pS for Li+) were smaller than those of alpha gamma channels (6.5 pS for Na+ and 10.8 pS for Li+); (d) the half-inhibition constant (Ki) of amiloride was 20-fold larger for alpha beta channels than for alpha gamma channels whereas the Ki of guanidinium was equal for both alpha beta and alpha gamma. To identify the domains in the channel subunits involved in amiloride binding, we constructed several chimeras that contained the amino terminus of the gamma subunit and the carboxy terminus of the beta subunit. A stretch of 15 amino acids, immediately before the second transmembrane domain of the beta subunit, was identified as the domain conferring lower amiloride affinity to the alpha beta channels. We provide evidence for the existence of two distinct binding sites for the amiloride molecule: one for the guanidium moiety and another for the pyrazine ring. At least two subunits alpha with beta or gamma contribute to these binding sites. Finally, we show that the most likely stoichiometry of alpha beta and alpha gamma channels is 1 alpha: 1 beta and 1 alpha: 1 gamma, respectively.

Lithium clearance in healthy humans: effects of sodium intake and diuretics.

In summary, our experiments clearly demonstrate that lithium reabsorption occurs by frusemide- and bumetanide-sensitive reabsorption, but we have failed to identify the mechanism(s) responsible for the lower CLi and FELi in salt-depletion. It is possible that some, as yet unknown, factor increases the activity of the Na, K, 2Cl cotransporter and, hence, increases lithium reabsorption in the thick ascending limb in salt-depleted subjects. However, it is equally possible that a fraction of proximal tubular reabsorption is inhibited by frusemide and bumetanide. If this is correct, CLi in humans are reasonable markers of proximal tubular function even in conditions of avid salt retention and in salt depletion, when fractional reabsorption of salt and water in the proximal tubules is enhanced.

Na-K-Cl cotransport in villus and crypt cells from rat duodenum.

The aim of the present study was to examine the possibility that Cl- uptake into both villus and crypt epithelial cells of rat duodenum occurs via an electroneutral Na-K-Cl coupled-transport mechanism. Sheets of villus cells and whole crypts were isolated using a Ca2+ chelation technique combined with continuous vibration at low temperatures. Structurally intact, viable epithelia from defined regions along the villus-crypt axis were produced. Uptake of 86Rb+ (as a proxy for K+) into both villus and crypt cells appeared to depend on a coupled process, as evidenced by the inhibition of 86Rb+ uptake by bumetanide and by the removal of either Na+ or Cl- from the bathing media. We report an improved method of isolation of viable enterocytes from defined regions along the villus-crypt axis. We demonstrate the presence of Na-K-Cl cotransport in both villus and crypt duodenal enterocytes.

Role of Ca2+/CaMK II in Ca(2+)-induced K+ channel inhibition in rat CCD principal cell.

The apical low-conductance K+ channel of rat cortical collecting duct (CCD) is inhibited by increased intracellular Ca2+ concentrations. This effect has been shown to be mediated at least in part by activation of protein kinase C (PKC). In the present study, we used the patch-clamp technique to examine the role of Ca2+/calmodulin-dependent protein kinase II (CaMK II) in mediating the Ca(2+)-induced inhibitory effect. In cell-attached patches of principal cells of rat tubules, clamping of intracellular Ca2+ concentration at 400 nM by using 1 microM ionomycin reduced channel activity to 26.5% of the control value. A further reduction in channel activity, to 8.8% of the control value, was observed following the addition of phorbol 12-myristate 13-acetate (PMA), an agent known to activate PKC. Pretreatment of cells with KN-62 (CaMK II inhibitor) or GF-109203X (PKC inhibitor) attenuated the inhibitory effect of Ca2+ on K+ channel activity (83.2 and 50.7% of the control value, respectively). Even in the presence of KN-62, addition of 10 microM PMA significantly decreased channel activity to 57.2% of the control value. The Ca(2+)-induced inhibition was completely abolished by simultaneous incubation with both KN-62 and GF-109203X. In inside-out patches, addition of 20 micrograms/ml CaMK II in the presence of a PKC inhibitor reduced channel activity to 66.2% of control values. It is concluded that CaMK II is involved in mediating the Ca(2+)-induced inhibition of the activity of the apical K+ channel of rat CCD.

A Cl- conductance sensitive to external pH in the apical membrane of rat duodenal enterocytes.

1. The pH dependence of a chloride conductance in the apical membrane of rat duodenal enterocytes was examined. 2. A stepwise reduction of both internal and external pH from 7.4 to 6.8 resulted in a significant stimulation of 36Cl flux driven by an inside-positive membrane potential. 3. A stepwise reduction in pH had no significant effect upon other parameters such as the initial rate of D-[3H]glucose or voltage-independent 36Cl uptake, suggesting a specific effect upon the chloride conductance. 4. The pH-dependent stimulation of 36Cl uptake exhibited saturation kinetics, with an apparent Vmax (maximum velocity) of 5.5 nmol (mg protein)-1 (4 s)-1 and an apparent Km (Michaelis-Menten constant) of 88 nM H+ ions. 5. To determine the site of action of protons upon the conductance the effect of asymmetrically reducing either the internal or external pH was examined. 6. A step reduction of extracellular pH from 7.8 to 6.8 significantly stimulated the rate of 36Cl uptake. In contrast, a step reduction of internal pH from 7.8 to 6.8 was without effect upon the rate of 36Cl uptake. 7. These results suggest that the chloride conductance on the apical membrane of rat duodenal enterocytes is allosterically regulated by protons at an external site.

Potassium channels in colonic crypts.

A characteristic feature of intestinal epithelia is their ability to secrete chloride (Cl-), a process that occurs mainly in intestinal crypts and is the critical transport event in secretory diarrhoea. Increased potassium (K+) channel activity in the basolateral membrane has an important role in the Cl- secretory process by hyperpolarising the cell and maintaining a favourable electrochemical driving force for Cl- exit at the apical membrane. We have shown, using patch-clamp techniques, that the basolateral membrane of human colonic crypt cells contains low conductance K+ channels that are voltage and calcium (Ca2+) sensitive and blocked by barium (Ba2+). These K+ channels are regulated by cytosolic cyclic adenosine monophosphate (cAMP) and Ca2+, intracellular second messengers that also stimulate Cl- secretion. This population of human intestinal K+ channels may be a target for the pharmacological control of Cl- secretory diarrhoea.

Properties and regulation of basolateral K+ channels in rat duodenal crypts.

1. Patch clamp recording techniques were used to study the properties of K+ channels in the basolateral membrane of rat duodenal crypts, and their regulation by Cl(-)-secretory agonists. 2. High conductance (84-99 pS) K+ channels were activated by 0.1 mM dibutyryl cAMP in 50% of cell-attached patches, while 0.1 mM carbachol had no effect on channel activity. High conductance K+ channels were voltage independent, Ca2+ insensitive, blocked by 5 mM Ba2+ or 20 mM tetraethylammonium chloride (TEA), and stimulated by 57% when intracellular pH was increased from 7.4 to 7.8. 3. In contrast, low conductance (19-28 pS) K+ channels were activated by 0.1 mM dibutyryl cAMP in 67% of cell-attached patches, while 0.1 mM carbachol activated channels in 100% of cell-attached patches. Low conductance K+ channels were voltage independent, Ca2+ sensitive and pH insensitive. Unlike the high conductance K+ channels, 5 mM Ba2+ had no effect on the activity of low conductance K+ channels, although 20 mM TEA decreased channel activity by 53%. 4. The results show that the basolateral membrane of rat duodenal crypts possesses two discrete populations of K+ channels that may have important roles in sustaining the small intestinal Cl(-)-secretory responses triggered by a variety of cAMP- and Ca(2+)-mediated agonists.

Regulation of ROMK1 K+ channel activity involves phosphorylation processes.

An inwardly rectifying, ATP-regulated K+ channel with a distinctive molecular architecture, ROMK1, was recently cloned from rat kidney. Using patch clamp techniques, we have investigated the regulation of ROMK1 with particular emphasis on phosphorylation/dephosphorylation processes. Spontaneous channel rundown occurred after excision of membrane patches into ATP-free bath solutions in the presence of Mg2+. Channel rundown was almost completely abolished after excision of patches into either Mg(2+)-free bathing solutions or after preincubation with the broad-spectrum phosphatase inhibitor, orthovanadate, in the presence of Mg2+. MgATP preincubation also inhibited channel rundown in a dose-dependent manner. In addition, the effect of the specific phosphatase inhibitors okadaic acid (1 microM) and calyculin A (1 microM) was also investigated. The presence of either okadaic acid or calyculin A failed to inhibit channel rundown. Taken together, these data suggest that rundown of ROMK1 involves a Mg(2+)-dependent dephosphorylation process. Channel activity was also partially restored after the addition of MgATP to the bath solution. Addition of exogenous cAMP-dependent protein kinase A (PKA) catalytic subunit led to a further increase in channel open probability. Addition of Na2ATP, in the absence of Mg2+, was ineffective, suggesting that restoration of channel activity is a Mg(2+)-dependent process. Addition of the specific PKA inhibitor, PKI, to the bath solution led to a partial, reversible inhibition in channel activity. Thus, PKA-dependent phosphorylation processes are involved in the modulation of channel activity. This observation is consistent with the presence of potential PKA phosphorylation sites on ROMK1.

Regulation of ATP-sensitive K+ channel by membrane-bound protein phosphatases in rat principal tubule cell.

The role of membrane-bound protein serine/threonine phosphatases (PP) in modulating the renal ATP-sensitive K+ (KATP) channel was examined using the patch-clamp technique in principal cells of rat cortical collecting duct. In the absence of ATP, channel activity rapidly (11.2 s) declines (channel "rundown") upon excision of the membrane patches into control bath solutions (1 mM Mg2+, Ca2+ free). Both orthovanadate (5 mM), a broad-spectrum inhibitor of phosphatases except for Ca(2+)-dependent PP (PP-2B), and okadaic acid (OA, 1 microM), a potent inhibitor of PP types 1 and 2A (PP-1 and PP-2A), significantly slowed channel rundown. Removal of Mg2+ from the bath also slowed the rundown process. Incubation of cells with OA in the absence of Mg2+ or with orthovanadate in ATP-free solution maintained channel activity at levels of approximately 70% of control values for 3 min after membrane excision. In contrast, Ca2+ (0.1 mM) and calmodulin (1 microM) in the presence of 1 mM Mg2+, a condition in which PP-2B is stimulated, had no significant effect on the channel activity that persisted in the presence of OA and orthovanadate. Application of exogenous PP-2A (1 U/ml) to the cytosolic side of membrane in inside-out patches significantly inhibited channel activity to 35.0% of control, but the inhibitory-effects of PP-1 (1 U/ml) and PP-2B (20 micrograms/ml) were minor. These results suggest that rundown of the renal KATP channel after membrane excision results mainly from dephosphorylation of the channel or an associated protein by membrane-bound phosphatases.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular site for nucleotide binding on an ATP-sensitive renal K+ channel (ROMK2).

ATP-sensitive, inwardly rectifying K+ channels are present in apical membranes of the distal nephron and play a major role in K+ recycling and secretion. The cloned renal K+ channel, ROMK1, is a candidate for the renal epithelial K+ channel, since it shares many functional characteristics with the native channel. Additionally, ROMK1 contains a putative carboxy-terminal ATP-binding site. Although ROMK1 channel activity could be reactivated by cytosolic Mg-ATP after rundown, the role of nucleotides in channel gating was less certain. We now show that an alternatively spliced transcript of the ROMK channel gene, ROMK2, which encodes a K+ channel with a truncated amino terminus, expresses an ATP-regulated and ATP-sensitive K+ channel (IKATP). Differences in the amino terminus of ROMK isoforms alters the sensitivity of the channel-gating mechanism to ATP. To test whether ATP sensitivity of renal IKATP is mediated by direct interaction of nucleotide, point mutation of specific residues within the ROMK2 phosphate loop (P-loop) were investigated. These either enhanced or attenuated the sensitivity to both activation and inhibition by Mg-ATP, thus demonstrating a direct interaction of nucleotide with the channel-forming polypeptide.

Partially active channels produced by PKA site mutation of the cloned renal K+ channel, ROMK2 (kir1.2).

The activity of the cloned renal K+ channel (ROMK2) is dependent on a balance between phosphorylation and dephosphorylation. There are only three protein kinase A (PKA) sites on ROMK2, with the phosphorylated residues being serine-25 (S25), serine-200 (S200), and serine-294 (S294) (Z.-C. Xu, Y. Yang, and S. C. Hebert. J. Biol. Chem. 271: 9313-9319, 1996). We previously mutated these sites from serine to alanine to study the contribution of each site to overall channel function. Here we have studied each of these single PKA site mutants using the single-channel configuration of the patch-clamp technique. Both COOH-terminal mutations at sites S200A and S294A showed a decreased open channel probability (Po), whereas the NH2-terminal mutation at site S25A showed no change in Po compared with wild-type ROMK2. The decrease in Po for the S200A and S294A mutants was caused by the additional presence of a long closed state. In contrast, the occurrence of the S25A channel was approximately 66% less, suggesting fewer active channels at the membrane. The S200A and S294A channels had different kinetics compared with wild-type ROMK2 channels, showing an increased occurrence of sublevels. Similar kinetics were observed when wild-type ROMK2 was excised and exposed to dephosphorylating conditions, indicating that these effects are specifically a property of the partially phosphorylated channel and not due to an unrelated effect of the mutation.

Aldosterone-induced apical Na+ and K+ conductances are located predominantly in surface cells in rat distal colon.

Aldosterone is a major regulator of Na(+)-absorptive and K(+)-secretory processes in the distal segment of mammalian colon. In this study, the distribution of aldosterone-sensitive cell types in isolated rat distal colon was determined using site-directed intracellular microelectrodes, specific Na(+)- and K(+)-channel blockers, and aldosterone-receptor binding techniques. Electrophysiological data indicated that aldosterone induced parallel apical membrane Na+ and K+ conductances, mainly in surface cells and to a significantly lesser degree in crypt cells. Scatchard analyses of aldosterone-receptor binding in cytosolic fractions revealed the maximum number of specific binding sites in whole mucosal homogenate and in the upper one-third and lower two-thirds of isolated crypt units to be 74.9 +/- 2.0, 59.8 +/- 2.4, and 59.3 +/- 3.2 fmol/mg protein, respectively, indicating the presence of aldosterone receptors in the crypt cell population. We conclude that in rat distal colon aldosterone-induced Na+ and K+ conductances (and by inference, electrogenic Na(+)-absorptive and K(+)-secretory processes) are located predominantly in the surface cell population and to a lesser extent in crypt cells, which also contain aldosterone receptors. This spectrum of aldosterone-induced Na+ and K+ conductances may reflect varying stages of differentiation along the surface cell-crypt cell axis.

A novel approach allows identification of K channels in the lateral membrane of rat CCD.

We have developed a novel approach to study K channels in the lateral membrane of principal cells (PC) in rat cortical collecting ducts (CCD). The technique consists of 1) exposing the CCD apical membrane, 2) removing the intercalated cells adjoining a PC by gentle suction through a pipette, and 3) applying patch-clamp technique to the lateral membrane of PC. Functional viability of the PC was confirmed by three indexes: 1) maintenance of physiological cell membrane potentials (-85 +/- 3 mV); 2) depolarization of the cell membrane potential with 1 mM Ba2+; and 3) hyperpolarization of the cell potential with 0.1 mM amiloride. Two types of K channels were identified: a low-conductance K channel and an intermediate-conductance K channel. In cell-attached patches the slope conductance of the low-conductance K channel was 27 pS and that of the intermediate-conductance K channel was 45 pS. The open probability (Po) of the 27-pS K channel was 0.81 +/- 0.02 and was not voltage dependent. In contrast, the Po of the 45-pS K channel was 0.23 +/- 0.01 at the spontaneous cell membrane potential and was increased by hyperpolarization. In addition, decrease of the bath pH from 7.4 to 6.7 reduced the 27-pS K channel current amplitude in a voltage-dependent manner, but the Po was not affected. Finally, two time constants were required to fit open- and closed-time histograms of both populations of K channels. Application of 1 mM Ba2+ completely blocked these K channels. We conclude that two types of K channel are present in the basolateral membrane of PC.

pH-dependent modulation of the cloned renal K+ channel, ROMK.

pH is an important modulator of the low-conductance ATP-sensitive K+ channel of the distal nephron. To examine the mechanism of interaction of protons with the channel-forming protein, we expressed the cloned renal K channel, ROMK (Kir1.x), in Xenopus oocytes and examined the response to varied concentrations of protons both in the presence and in the absence of ATP. Initial experiments were performed on inside-out patches in the absence of ATP in Mg2+-free solution, which prevents channel rundown. A steep sigmoidal relationship was shown between bath pH and ROMK1 or ROMK2 channel function with intracellular acidification reducing channel activity. We calculated values for pK = 7.18 and 7.04 and Hill coefficients = 3.1 and 3.3, for ROMK1 and ROMK2, respectively. Intracellular acidification (pH 7.2) also increased the Mg-ATP binding affinity of ROMK2, resulting in a leftward shift of the relationship between ATP concentration and the reduction in channel activity. The K1/2 for Mg-ATP decreased from 2.4 mM at pH 7.4 to approximately 0.5 mM at pH 7.2. Mutation of lysine-61 to methionine in ROMK2, which abolishes pH sensitivity, modulated but did not eliminate the effect of pH on ATP inhibition of channel activity. We previously demonstrated that the putative phosphate loop in the carboxy terminus of ROMK2 is involved in ATP binding and channel inhibition [C. M. McNicholas, Y. Yang, G. Giebisch, and S. C. Hebert. Am. J. Physiol. 271 (Renal Fluid Electrolyte Physiol. 40): F275-F285, 1996]. Conceivably, therefore, protonation of the histidine residue within this region could alter net charge (i.e., positive shift) and increase affinity for the negatively charged nucleotide.

Sensitivity of a renal K+ channel (ROMK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator.

We demonstrate here that coexpression of ROMK2, an inwardly rectifying ATP-sensitive renal K+ channel (IKATP) with cystic fibrosis transmembrane regulator (CFTR) significantly enhances the sensitivity of ROMK2 to the sulfonylurea compound glibenclamide. When expressed alone, ROMK2 is relatively insensitive to glibenclamide. The interaction between ROMK2, CFTR, and glibenclamide is modulated by altering the phosphorylation state of either ROMK2, CFTR, or an associated protein, as exogenous MgATP and the catalytic subunit of protein kinase A significantly attenuate the inhibitory effect of glibenclamide on ROMK2. Thus CFTR, which has been demonstrated to interact with both Na+ and Cl- channels in airway epithelium, modulates the function of renal ROMK2 K+ channels.

Primary structure and functional expression of a cGMP-gated potassium channel.

Cyclic nucleotides modulate potassium (K) channel activity in many cells and are thought to act indirectly by inducing channel protein phosphorylation. Herein we report the isolation from rabbit of a gene encoding a K channel (Kcn1) that is specifically activated by cGMP and not by cAMP. Analysis of the deduced amino acid sequence (725 amino acids) indicates that, in addition to a core region that is highly homologous to Shaker K channels, Kcn1 also contains a cysteine-rich region similar to that of ligand-gated ion channels and a cyclic nucleotide-binding region. Northern blot analysis detects gene expression in kidney, aorta, and brain. Kcn1 represents a class of K channels that may be specifically regulated by cGMP and could play an important role in mediating the effects of substances, such as nitric oxide, that increase intracellular cGMP.

A functional CFTR-NBF1 is required for ROMK2-CFTR interaction.

In a previous study on inside-out patches of Xenopus oocytes, we demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR) enhances the glibenclamide sensitivity of a coexpressed inwardly rectifying K+ channel, ROMK2 (C. M. McNicholas, W. B. Guggino, E. M. Schwiebert, S. C. Hebert, G. Giebisch, and M. E. Egan. Proc. Natl. Acad. Sci. USA 93: 8083-8088, 1996). In the present study, we used the two-microelectrode voltage-clamp technique to measure whole cell K+ currents in Xenopus oocytes, and we further characterized the enhanced sensitivity of ROMK2 to glibenclamide by CFTR. Glibenclamide inhibited K+ currents by 56% in oocytes expressing both ROMK2 and CFTR but only 11% in oocytes expressing ROMK2 alone. To examine the role of the first nucleotide binding fold (NBF1) of CFTR in the ROMK2-CFTR interaction, we studied the glibenclamide sensitivity of ROMK2 when coexpressed with CFTR constructs containing mutations in or around the NBF1 domain. In oocytes coinjected with ROMK2 and a truncated construct of CFTR with an intact NBF1 (CFTR-K593X), glibenclamide inhibited K+ currents by 46%. However, in oocytes coinjected with ROMK2 and a CFTR mutant truncated immediately before NBF1 (CFTR-K370X), glibenclamide inhibited K+ currents by 12%. Also, oocytes expressing both ROMK2 and CFTR mutants with naturally occurring NBF1 point mutations, CFTR-G551D or CFTR-A455E, display glibenclamide-inhibitable K+ currents of only 14 and 25%, respectively. Because CFTR mutations that alter the NBF1 domain reduce the glibenclamide sensitivity of the coexpressed ROMK2 channel, we conclude that the NBF1 motif is necessary for the CFTR-ROMK2 interaction that confers sulfonylurea sensitivity.

Isolation and structure elucidation of Chlorofusin, a novel p53-MDM2 antagonist from a Fusarium sp.

Wild-type p53 plays a crucial role in the prevention of cancer. Since dysfunction of p53 can be caused by increased levels of the protein MDM2, small molecules which antagonize the interaction between these two proteins have potential in cancer therapy. The discovery and structure determination of a fungal metabolite, chlorofusin, which antagonizes the p53/MDM2 interaction are reported.