High risk of drug-resistant tuberculosis when first-line therapy fails in a high HIV prevalence setting.

SETTING: Lesotho national multidrug-resistant tuberculosis (MDR-TB) program. OBJECTIVE: To determine the prevalence of drug-resistant TB (DR-TB) among patients registered for MDR-TB treatment after failure or suspected failure of the standard 6-month regimen for new TB patients (Category I). DESIGN: We conducted a retrospective cohort study of patients registered for MDR-TB treatment following failure or suspected failure of Category I. RESULTS: A total of 76 patients were included in the analysis, including 51 Category I treatment failures and 25 suspected Category I treatment failures. The prevalence of resistance to any drug was 92% among the treatment failures and 72% among the suspected failures. The proportion of MDR-TB was respectively 78% and 28% among the treatment failures and suspected failures. Among the subgroup of human immunodeficiency virus (HIV) positive patients, the proportion of MDR-TB was 84% among failures and 23% among suspected failures. CONCLUSION: DR-TB and MDR-TB were common among patients in whom Category I failed. Early initiation of empiric second-line anti-tuberculosis treatment while awaiting culture and drug susceptibility testing (DST) results should be considered for HIV-negative and -positive patients who have failed first-line anti-tuberculosis treatment; patients suspected to be failing a first-line regimen should undergo DST at the end of the intensive phase.

High rate of hypothyroidism among patients treated for multidrug-resistant tuberculosis in Lesotho.

BACKGROUND: Hypothyroidism is a known side effect of treatment for multidrug-resistant tuberculosis (MDR-TB), but it is considered to be rare. Hypothyroidism has vague and non-specific symptoms, and can be easily missed by clinicians. OBJECTIVE: To report the high rate of hypothyroidism in a cohort of MDR-TB patients in Lesotho and to describe our approach to diagnosis and management. DESIGN: A retrospective study of 212 patients who initiated treatment for MDR-TB in Lesotho between 27 July 2007 and 24 March 2009 was performed. RESULTS: Among 186 patients screened, 129 (69%) had hypothyroidism, defined as at least one documented thyroid-stimulating hormone (TSH) result > 10.0 mIU/l; 100 (54%) patients had a maximum TSH > 20.0 mIU/l. At 93 days after starting MDR-TB treatment, half of the patients had developed hypothyroidism. CONCLUSION: Hypothyroidism may be more common during MDR-TB treatment than previously recognized. Screening all patients, even those without symptoms, for hypothyroidism within 2-3 months of starting MDR-TB treatment should be considered until prospective studies can inform screening guidelines.

Temperature-sensitive differential affinity of TRAIL for its receptors. DR5 is the highest affinity receptor.

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines which induces apoptotic cell death in a variety of tumor cell lines. It mediates its apoptotic effects through one of two receptors, DR4 and DR5, which are members of of the TNF receptor family, and whose cytoplasmic regions contain death domains. In addition, TRAIL also binds to 3 "decoy" receptors, DcR2, a receptor with a truncated death domain, DcR1, a glycosylphosphatidylinositol-anchored receptor, and OPG a secreted protein which is also known to bind to another member of the TNF family, RANKL. However, although apoptosis depends on the expression of one or both of the death domain containing receptors DR4 and/or DR5, resistance to TRAIL-induced apoptosis does not correlate with the expression of the "decoy" receptors. Previously, TRAIL has been described to bind to all its receptors with equivalent high affinities. In the present work, we show, by isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay, that the rank order of affinities of TRAIL for the recombinant soluble forms of its receptors is strongly temperature dependent. Although DR4, DR5, DcR1, and OPG show similar affinities for TRAIL at 4 degrees C, their rank-ordered affinities are substantially different at 37 degrees C, with DR5 having the highest affinity (K(D)

A G protein-coupled receptor for UDP-glucose.

Uridine 5'-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist. Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.

Acquisition of sensitivity of stress-activated protein kinases to the p38 inhibitor, SB 203580, by alteration of one or more amino acids within the ATP binding pocket.

Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase compete with ATP for binding. Mutation of 23 residues in the ATP pocket indicated that several residues which affected binding of pyridinyl imidazole photoaffinity cross-linker 125I-SB 206718 did not affect kinase activity, and vice versa, suggesting that pyridinyl imidazoles bind p38 differently than ATP. Two close homologues of p38, SAPK3 and SAPK4, are not inhibited by SB 203580 and differ from p38 by three amino acids near the hinge of the ATP pocket. Substitution of the three amino acids in p38 by those in SAPK3/4 (Thr-106, His-107, and Leu-108 to Met, Pro, and Phe) resulted in decreased 125I-SB 206718 cross-linking and loss of inhibition by SB 203580. Substitution of just Thr-106 by Met resulted in incomplete loss of inhibition. Conversely, substitution of the three amino acids of p38 into SAPK3, SAPK4, or the more distantly related JNK1 resulted in inhibition by SB 203580, whereas mutation of just Met-106 to Thr resulted in weaker inhibition. These results indicate that these three amino acids can confer specificity and sensitivity to SB 203580 for at least two different classes of MAPKs.

Role of conserved histidines in catalytic activity and inhibitor binding of human recombinant phosphodiesterase 4A.

To identify critical amino acids within the central conserved region of recombinant human cAMP-specific phosphodiesterase 4 subtype A (rhPDE4A), we engineered the expression of point mutants in a fully active rhPDE4A/Met201-886. When histidine residues at positions 433, 437, 473, and 477, which are highly conserved among all PDE families, were changed independently to serine residues, cAMP hydrolyzing activities were substantially reduced or abolished. The ability of these mutants to bind prototypical PDE4 inhibitors [3H]-(R)-rolipram or [3H]RP 73401 was also decreased in parallel with the loss of catalytic activity. The parallel loss of catalytic activity and inhibitor binding suggests that these changes resulted from non-localized perturbations in the structure of the enzyme. More interesting results were obtained when histidine residues at positions 505 and 506 were changed independently to aspar agines. The K(m) value for cAMP increased 3-fold in H505N (K(m) = 11 +/- 3 microM) and 11-fold in H506N (K(m) = 44 +/- 6 microM) compared with the wild-type protein (K(m) = 4 +/- 1 microM). These mutant proteins bound [3H]-(R)-rolipram and [3H]RP 73401 with K(d) values of 1.8 +/- 0.4 and 0.3 +/- 0.1 nM, respectively, for H505N, and 3.9 +/- 0.9 and 0.5 +/- 0.1 nM, respectively, for H506N. These values are nearly identical to those obtained with the wild-type rhPDE4A/Met201-886. In contrast, the IC50 values for cAMP competition with either [3H]-(R)-rolipram or [3H]RP 73401 binding increased approximately 2-fold in H505N and approximately 13-fold in H506N compared with the wild type protein. These increases are virtually identical to the changes in the K(m) value for cAMP in these mutants. We conclude that His506 and, perhaps, His505 are involved in binding of cAMP to PDE4A/Met201-886 but not in binding of PDE4-selective inhibitors.

Autocatalytic activation of human cathepsin K.

The in vitro activation of the recombinant purified human cathepsin K (EC 3.4.22.38) was examined by mutagenesis. Cathepsin K was expressed as a secreted proenzyme using baculovirus-infected Sf21 insect cells. Spontaneous in vitro activation of procathepsin K occurred at pH 4 and was catalyzed by exogenous mature cathepsin K. Three intermediates were identified as resulting from cleavages after Glu19, Ser98, and Glu110. The mature enzyme was composed of mixture of enzymes with N termini of Gly113, Arg114, and Ala115 with varying ratios depending on the preparation. Molecular weight determinations were consistent with the absence of carbohydrate in the mature protein, while electrospray mass spectroscopy indicated that six of the eight cysteine residues were in disulfide linkage, and that the protein had Met329 as the C-terminal residue. A mutant was constructed in which the active site Cys139 was changed to Ser. [Ser139,Ala163]Procathepsin K (containing mutation C139S,S163A) failed to spontaneously process and was only partially processed in the presence of 1% exogenous wild-type mature cathepsin K forming intermediates, which were identical to those observed in the activation of wild-type. [Ser139,Ala163]Procathepsin K could be fully processed to mature enzyme by including one equivalent of wild-type procathepsin K in the activation mixture. These results indicated that in vitro activation of the procathepsin K was an autocatalytic process.

Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase bind in the ATP site.

The site of action of a series of pyridinyl imidazole compounds that are selective inhibitors of p38 mitogen-activated protein kinase in vitro and block proinflammatory cytokine production in vivo has been determined. Using Edman sequencing, 125I-SB206718 was shown to cross-link to the nonphosphorylated Escherichia coli-expressed p38 kinase at Thr175, which is proximal to the ATP binding site. Titration calorimetric studies with E. coli-expressed p38 kinase showed that SB203580 bound with a stoichiometry of 1:1 and that binding was blocked by preincubation of p38 kinase with the ATP analogue, FSBA (5'-[p-(fluorosulfonyl)benzoyl]adenosine), which covalently modifies the ATP binding site. The intrinsic ATPase activity of the nonphosphorylated enzyme was inhibited by SB203580 with a Km of 9.6 mM. Kinetic studies of active, phosphorylated yeast-expressed p38 kinase using a peptide substrate showed that SB203580 was competitive with ATP with a Ki of 21 nM and that kinase inhibition correlated with binding and biological activity. Mutagenesis indicated that binding of 125I-SB206718 was dependent on the catalytic residues K53 and D168 in the ATP pocket. These findings indicate that the pyridinyl imidazoles act in vivo by inhibiting p38 kinase activity through competition with ATP and that their selectivity is probably determined by differences in nonconserved regions within or near the ATP binding pocket.

Mapping the functional domains of human recombinant phosphodiesterase 4A: structural requirements for catalytic activity and rolipram binding.

To identify functional domains of the 886-amino acid human recombinant cAMP-specific phosphodiesterase (PDE) subtype A (rhPDE4A), we engineered the expression of seven mutant proteins containing both NH2- and COOH-terminal truncations. The level of rhPDE4A protein expression in yeast was monitored by immunoblotting using enzyme-specific antisera. Biochemical profiles of the mutant proteins were compared with those of the full-length protein or a fully active truncated form of the enzyme (rhPDE4A Met265-886), lacking the first 264 amino acids. The smallest catalytically active fragment generated was Met332-722, which at 45 kDa is less than half the mass of the full-length enzyme (approximately 110 kDa) but spans the most highly conserved region of the PDE superfamily. Two prototypical PDE4 inhibitors, rolipram and RP 73401, inhibited cAMP hydrolyzing activity of all truncated forms of the enzyme, with IC50 values of 70-2000 nM and 0.2-0.6 nM, respectively. [3H](R)-Rolipram bound to two sites on Met265-886, a high affinity site (Kd1 = 0.7 +/- 0.3 nM) and a low affinity site (Kd2 = 34 +/- 10 nM). Interestingly, [3H](R)-rolipram failed to bind to Met332-886 with high affinity, indicating that high affinity binding is not required for inhibition of enzyme activity. Low affinity rolipram binding was still present in Met332-886 (Kd = 101 +/- 7 nM). In contrast to [3H](R)-rolipram, [3H]RP 73401 bound to a single class of high affinity sites on Met265-886 (Kd = 0.4 +/- 0.1 nM). Further truncation of the enzyme to Met332-886 had no effect on [3H]RP 73401 binding (Kd = 0.2 +/- 0.03 nM). We conclude that the catalytic center of rhPDE4A lies between amino acids 332 and 722. Furthermore, amino acids 265-332 may form a high affinity binding site for rolipram that is outside of the catalytic domain. As a more likely alternative, these amino acids may not form a distinct binding site but instead may be required for the recombinant enzyme to assume a conformation that binds rolipram at the catalytic domain with a high affinity.

Human platelet cGI-PDE: expression in yeast and localization of the catalytic domain by deletion mutagenesis.

Cyclic adenosine monophosphate (cAMP) is an important modulator of platelet responses to agonists. Cyclic nucleotide phosphodiesterase (PDE) controls intracellular cAMP concentrations by hydrolyzing it to AMP. The major PDE activity in platelets is PDE3A (cyclic guanosine monophosphate [cGMP]-inhibited PDE). To obtain structural information on platelet PDE3A, we cloned the enzyme cDNA from a human erythroleukemia cell (HEL) library since the cell line expresses many platelet proteins. This clone consists of 87% of the full-length human myocardial PDE3A cDNA, spanning from nucleotides 456 to 4606, and is identical in sequence. The nucleotide coding for the N terminal 179 amino acid sequence (nt 1-536) as well as four other cDNAs (nt 1459-1632, nt 1765-1986, nt 2152-2538, and nt 2978-3375) obtained by RT-PCR of platelet RNA are also identical to the myocardial sequences, indicating that the HEL, myocardial, and platelet PDE3As are the same. Northern blot analysis of HEL cell RNA detected two mRNAs of 7.5 and 4.4 kb. Four new deletion mutants are reported. PDE 3A delta 1 and PDE 3A delta 2, encoding amino acids 665 to 1141 and amino acids 679 to 1141, respectively, were expressed in a PDE-deficient yeast. They displayed PDE activities of 172 and 79 pmol/mg/min, respectively. PDE 3A delta 3 and PDE 3A delta 4, encoding amino acids 686 to 1141 and 700 to 1141, had no detectable PDE activity. All mutant proteins were expressed as determined by Western blot analysis. These findings localize the PDE3A catalytic domain to within amino acid residues 679 to 1141.

Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase.

CSBP p38 is a mitogen-activated protein kinase that is activated in response to stress, endotoxin, interleukin 1, and tumor necrosis factor. Using a catalytically inactive mutant (D168A) of human CSBP2 as the bait in a yeast two-hybrid screen, we have identified and cloned a novel kinase which shares approximately 70% amino acid identity to mitogen-activated protein kinase-activated protein kinase (MAPKAP kinase)-2, and thus was designated MAPKAP kinase-3. The binding of CSBP to MAPKAP kinase-3 was confirmed in vitro by the precipitation of epitope-tagged CSBP1, CSBP2, and CSBP2(D168A) and endogenous CSBP from mammalian cells by a bacterially expressed GST-MAPKAP kinase-3 fusion protein and in vivo by co-precipitation of the epitope-tagged proteins co-expressed in HeLa cells. MAPKAP kinase-3 was phosphorylated by both CSBP1 and CSBP2 and was then able to phosphorylate HSP27 in vitro. Treatment of HeLa cells with sorbitol or TNF resulted in activation of CSBP and MAPKAP kinase-3 and activation of MAPKAP kinase-3 could be blocked by preincubation of cells with SB203580, a specific inhibitor of CSBP kinase activity. These data suggest that MAPKAP kinase-3 is activated by stress and cytokines and is a novel substrate of CSBP both in vitro and in vivo.

Prolonged beta adrenoceptor stimulation up-regulates cAMP phosphodiesterase activity in human monocytes by increasing mRNA and protein for phosphodiesterases 4A and 4B.

Human peripheral blood monocytes were treated for 4 h with a combination of the beta-agonist salbutamol (3 microM) and the low-Km cAMP-specific phosphodiesterase (PDE4) inhibitor rolipram (30 microM) to produce a prolonged elevation of cAMP and consequent increase in PDE activity. After this treatment, isozyme-selective PDE inhibitors were used to characterize the cAMP PDE profiles of high-speed supernatants before and after DEAE-Sepharose column chromatography. These experiments, in which total soluble PDE activity was increased by 58%, showed that the increased PDE activity is due to up-regulation of PDE4 and that at least two of the four subtypes are up-regulated. Experiments in whole cells demonstrated that this relatively modest increase in PDE4 activity has significant functional consequences, reducing cAMP accumulation in response to both PGE2 and lower, though not maximal, concentrations of rolipram. Further characterization of PDE4 subtype expression in control and treated monocytes, using polymerase chain reaction and Western blotting with subtype-specific peptide antibodies, showed that resting monocytes express both mRNA and protein for PDE4A, PDE4B and PDE4D. The amount of message for PDE4A and PDE4B appeared to increase upon up-regulation, whereas mRNA for PDE4D was not detected in treated cells. Western blots showed increases in the amount of protein for both PDE4A and PDE4B after treatment. We conclude that the PDE4 subtypes are differentially regulated upon prolonged exposure to elevated cAMP, with the consequence that the PDE4 profiles of control and treated cells differ not only in total activity but also in the relative proportions of the subtypes represented.

Human mitogen-activated protein kinase CSBP1, but not CSBP2, complements a hog1 deletion in yeast.

CSBP1 and CSBP2 are human homologues of the Saccharomyces cerevisiae Hog1 mitogen-activated protein kinase which is required for growth in high osmolarity media. Expression of CSBP1, but not CSBP2, complemented a hog1 delta phenotype. A CSBP2 mutant (A34V) that complements hog1 delta was isolated and found to have approximately 3-fold lower kinase activity than the wild-type CSBP2. Further analysis revealed that both the kinase activity and tyrosine phosphorylation of CSBP1 and CSBP2 (A34V) is regulated by salt. In contrast, wild-type CSBP2 is constitutively active but dependent on the upstream kinase, Pbs2. Mutagenesis studies showed that reduction or elimination of CSBP2 kinase activity restores salt responsiveness as measured by tyrosine phosphorylation suggesting that too high a level of kinase activity can result in desensitization of the host cell and inability to grow in high salt.

Enhanced recovery of Haemophilus ducreyi from clinical specimens by incubation at 33 versus 35 degrees C.

Isolation rates of Haemophilus ducreyi from cases of chancroid are low. Experts recommend that isolation media be incubated at 33 to 35 degrees C, but the possible effect of this temperature range on the recovery of H. ducreyi has not been evaluated. We inoculated two sets of agar plates with material from genital ulcers and incubated one set at 33 degrees C and one at 35 degrees C; incubation at 33 degrees C identified 21% more cases than did incubation at 35 degrees C (109 versus 85 cases, respectively, of the 116 cases from which an isolation was made; P < 0.01).

A pilot study to evaluate the development of resistance to nevirapine in asymptomatic human immunodeficiency virus-infected patients with CD4 cell counts of > 500/mm3: AIDS Clinical Trials Group Protocol 208.

Treatment of human immunodeficiency virus (HIV) infection with nevirapine in patients with < 400 CD4 cells/mm3 rapidly selects for virus with reduced susceptibility to nevirapine. To test whether resistance would develop less quickly in patients with a lower virus burden, nevirapine was studied in asymptomatic patients with > 500 CD4 cells/mm3. With 400 mg of nevirapine daily, the median reduction in HIV RNA was 0.51 log10 copies/mL, and all isolates recovered by 12 weeks were resistant to nevirapine. As in patients with lower CD4 cell counts, some patients experienced sustained reduction in plasma HIV RNA despite the presence of resistant virus. These results suggest that lower levels of HIV RNA and immunosuppression did not retard the rate of emergence of nevirapine-resistant virus; also, a polymerase chain reaction-based HIV RNA assay is sufficiently sensitive to evaluate the antiviral effect of a drug in patients with > 500 CD4 cells/mm3.

Characterization of two human cAMP-specific phosphodiesterase subtypes expressed in baculovirus-infected insect cells.

Recombinant baculoviruses were constructed to express cDNAs encoding two distinct subtypes of human cAMP-specific phosphodiesterase (hPDE4A and hPDE4B). Infection of Spodoptera frugiperda insect cells with the appropriate recombinant baculoviruses resulted in high level production of biologically-active protein as measured by enzymatic activity and immunoblotting using subtype-specific anti-hPDE4 antisera. Both recombinant proteins showed catalytic activity with a low Km (approximately 3 microM) for cAMP (with no cGMP hydrolyzing activity) and were inhibited by R-rolipram with apparent Kis of 0.38 and 0.25 microM, respectively. The recombinant enzymes also contained saturable, stereoselective and high-affinity rolipram-binding sites (Kd approximately 2 nM). Thus, insect cell-derived hPDE4s possess kinetic properties analogous to native enzymes as well as to recombinant enzymes produced in yeast.

Phase I/II evaluation of nevirapine alone and in combination with zidovudine for infection with human immunodeficiency virus.

In these Phase I/II open-label clinical trials, 62 persons with human immunodeficiency virus type 1 (HIV-1) infection and CD4+ cell counts < 400/mm3 received nevirapine at doses of 12.5, 50, and 200 mg/day, alone or in combination with zidovudine, 200 mg q8h. Nevirapine was well tolerated in the doses tested. Mean steady-state trough levels were 0.23, 1.1, and 1.9 micrograms/ml for the 12.5, 50, and 200 mg/day doses, respectively. Early suppression of p24 antigen levels and increase in CD4+ cell count were reversed following rapid emergence of virus less susceptible to nevirapine. Resistant strains were isolated from all participants by 8 weeks. Nevertheless, reduction of p24 antigen levels to < 50% of baseline values persisted for 12 weeks or more in four of seven persons who received 200 mg nevirapine/day in combination with zidovudine: these individuals had been antigenemic on long-term zidovudine therapy. This study demonstrates a direct relationship between drug resistance and effects on surrogate markers in HIV-1 infection.

The yeast FKS1 gene encodes a novel membrane protein, mutations in which confer FK506 and cyclosporin A hypersensitivity and calcineurin-dependent growth.

FK506 and cyclosporin A (CsA) are potent immunosuppressive agents that display antifungal activity. They act by blocking a Ca(2+)-dependent signal transduction pathway leading to interleukin-2 transcription. Each drug forms a complex with its cognate cytosolic immunophilin receptor (i.e., FKBP12-FK506 and cyclophilin-CsA) which acts to inhibit the Ca2+/calmodulin-dependent protein phosphatase 2B, or calcineurin (CN). We and others have defined the Saccharomyces cerevisiae FKS1 gene by recessive mutations resulting in 100-1000-fold hypersensitivity to FK506 and CsA (as compared to wild type), but which do not affect sensitivity to a variety of other antifungal drugs. The fks1 mutant also exhibits a slow-growth phenotype that can be partially alleviated by exogenously added Ca2+ [Parent et al., J. Gen. Microbiol. 139 (1993) 2973-2984]. We have cloned FKS1 by complementation of the drug-hypersensitive phenotype. It contains a long open reading frame encoding a novel 1876-amino-acid (215 kDa) protein which shows no similarity to CN or to other protein phosphatases. The FKS1 protein is predicted to contain 10 to 12 transmembrane domains with a structure resembling integral membrane transporter proteins. Genomic disruption experiments indicate that FKS1 encodes a nonessential function; fks1::LEU2 cells exhibit the same growth and recessive drug-hypersensitive phenotypes observed in the original fks1 mutants. Furthermore, the fks1::LEU2 allele is synthetically lethal in combination with disruptions of both of the nonessential genes encoding the alternative forms of the catalytic A subunit of CN (CNA1 and CNA2). These data suggest that FKS1 provides a unique cellular function which, when absent, increases FK506 and CsA sensitivity by making the CNs (or a CN-dependent function) essential.

Interaction between FKBP12-rapamycin and TOR involves a conserved serine residue.

The yeast TOR1 and TOR2 proteins were previously discovered as putative targets of the immunosuppressive drug rapamycin. Although their cellular function is unknown, they are predicted to be at least 215 kDa in size and possess a C-terminal phosphatidylinositol (PI) kinase-related domain. We previously identified a conserved Ser residue, within the PI kinase-related domain of both yeast TOR proteins (Ser1972 in TOR1; Ser1975 in TOR2), as being the site of missense mutations conferring dominant rapamycin resistance. The Ser1972/1975 residue of yeast TOR is conserved in mammalian TOR homologs. One possibility is that this residue is critical for a direct interaction between TOR and the FKBP12-rapamycin complex. There is very recent biochemical evidence for an interaction between mammalian TOR and FKBP12-rapamycin (Brown, E. J., Albers, M. W., Shin, T. B., Ichikawa, K., Keith, C. T., Lane, W. S., and Schreiber, S. L. (1994) Nature 369, 756-758; Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P., and Snyder, S. H. (1994) Cell 78, 35-43). Using the yeast two-hybrid system, we now have obtained genetic proof of a physical interaction between FKBP12-rapamycin and TOR and have demonstrated that this interaction requires the conserved Ser residue. We have found that a small fragment of wild-type yeast TOR2 spanning Ser1975 is capable of interacting with human FKBP12 in the presence of rapamycin, whereas an Arg1975 mutant fails to interact. This effect is dependent upon rapamycin and is antagonized by FK506.

A protein kinase involved in the regulation of inflammatory cytokine biosynthesis.

Production of interleukin-1 and tumour necrosis factor from stimulated human monocytes is inhibited by a new series of pyridinyl-imidazole compounds. Using radiolabelled and radio-photoaffinity-labelled chemical probes, the target of these compounds was identified as a pair of closely related mitogen-activated protein kinase homologues, termed CSBPs. Binding of the pyridinyl-imidazole compounds inhibited CSBP kinase activity and could be directly correlated with their ability to inhibit cytokine production, suggesting that the CSBPs are critical for cytokine production.