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The discovery of compounds and proteins from plants has greatly contributed to modern medicine. Vernonia amygdalina Del. (Compositae) is used by humans and primates for a variety of conditions including parasitic infection. This paper describes the serendipitous discovery that V. amygdalina extract was able to bind to, and functionally inhibit, active TGFβ1. The binding agent was isolated and identified as chlorophyll a-b binding protein AB96. Given that active TGFβ1 contributes to the pathology of many infectious diseases, inhibiting these processes may explain some of the benefits associated with the ingestion of this species. This is the first plant-derived cytokine-neutralizing protein to be described and paves the way for further such discoveries.

A series of 1-methyl-1H-pyrazole-5-carboxamides were synthesized as potent inhibitors of the parasitic nematode of sheep, Haemonchus contortus. These compounds did not show overt cytotoxicity to a range of mammalian cell lines under standard in vitro culture conditions, had high selectivity indices, and were progressed to an acute toxicity study in a rodent model. Strikingly, acute toxicity was observed in mice. Experiments measuring cellular respiration showed a dose-dependent inhibition of mitochondrial respiration. Under these conditions, potent cytotoxicity was observed for these compounds in rat hepatocytes suggesting that the potent acute mammalian toxicity of this chemotype is most likely associated with respiratory inhibition. In contrast, parasite toxicity was not correlated to acute toxicity or cytotoxicity in respiring cells. This paper highlights the importance of identifying an appropriate in vitro predictor of in vivo toxicity early on in the drug discovery pipeline, in particular assessment for in vitro mitochondrial toxicity.

Despite repeated malaria infection, individuals living in areas where malaria is endemic remain vulnerable to reinfection. The Janus kinase (JAK1/2) inhibitor ruxolitinib could potentially disrupt the parasite-induced dysfunctional immune response when administered with antimalarial therapy. This randomized, single-blind, placebo-controlled, single-center phase 1 trial investigated the safety, tolerability, and pharmacokinetic and pharmacodynamic profile of ruxolitinib and the approved antimalarial artemether-lumefantrine in combination. Ruxolitinib pharmacodynamics were assessed by inhibition of phosphorylation of signal transducer and activator of transcription 3 (pSTAT3). Eight healthy male and female participants ages 18 to 55 years were randomized to either ruxolitinib (20 mg) (n = 6) or placebo (n = 2) administered 2 h after artemether-lumefantrine (80/480 mg) twice daily for 3 days. Mild adverse events occurred in six participants (four ruxolitinib; two placebo). The combination of artemether-lumefantrine and ruxolitinib was well tolerated, with adverse events and pharmacokinetics consistent with the known profiles of both drugs. The incidence of adverse events and artemether, dihydroartemisinin (the major active metabolite of artemether), and lumefantrine exposure were not affected by ruxolitinib coadministration. Ruxolitinib coadministration resulted in a 3-fold-greater pSTAT3 inhibition compared to placebo (geometric mean ratio = 3.01 [90% confidence interval = 2.14 to 4.24]), with a direct and predictable relationship between ruxolitinib plasma concentrations and %pSTAT3 inhibition. This study supports the investigation of the combination of artemether-lumefantrine and ruxolitinib in healthy volunteers infected with Plasmodium falciparum malaria. (This study has been registered at ClinicalTrials.gov under registration no. NCT04456634.)

A phenotypic screen of two different libraries of small molecules against the motility and development of the parasitic nematode Haemonchus contortus led to the identification of two 1-methyl-1 H-pyrazole-5-carboxamide derivatives. Medicinal chemistry optimization targeted modifications of the left-hand side, middle section, and right-hand side of the hybrid structure of these two hits to elucidate the structure-activity relationship (SAR). Initial SAR around these hits allowed for the iterative and directed assembly of a focused set of 30 analogues of their hybrid structure. Compounds 10, 17, 20, and 22 were identified as the most potent compounds, inhibiting the development of the fourth larval (L4) stage of H. contortus at sub-nanomolar potencies while displaying strong selectivity toward the parasite when tested in vitro against the human MCF10A cell line. In addition, compounds 9 and 27 showed promising activity against a panel of other parasitic nematodes, including hookworms and whipworms.

Recently, we have discovered that the registered pesticide, tolfenpyrad, unexpectedly and potently inhibits the development of the L4 larval stage of the parasitic nematode Haemonchus contortus with an IC50 value of 0.03 μM while displaying good selectivity, with an IC50 of 37.9 μM for cytotoxicity. As a promising molecular template for medicinal chemistry optimization, we undertook anthelmintic structure–activity relationships for this chemical. Modifications of the left-hand side (LHS), right-hand side (RHS), and middle section of the scaffold were explored to produce a set of 57 analogues. Analogues 25, 29, and 33 were shown to be the most potent compounds of the series, with IC50 values at a subnanomolar level of potency against the chemotherapeutically relevant fourth larval (L4) stage of H. contortus. Selected compounds from the series also showed promising activity against a panel of other different parasitic nematodes, such as hookworms and whipworms.

The discovery of compounds and proteins from plants has greatly contributed to modern medicine, especially in malaria where both quinine and artemisinin have been the cornerstone of therapeutics. Here we describe the first known plant-derived cytokine binding agent. Chlorophyll a-b binding protein AB96, present to varying levels in the leaves of all chlorophyll-containing plants, binds to active TGFβ1. Active TGFβ1 contributes to the pathology of many infectious and neoplastic diseases and therefore, by inhibiting these processes, chlorophyll a-b binding protein opens up new approaches with therapeutic potential.

Background The resistance of melanoma cells to cisplatin restricts its clinical use. Therefore, the search for novel tumor inhibitors and effective combination treatments that sensitize tumor cells to this drug are still needed. We purified macrovipecetin, a novel heterodimeric C-type lectin, from Macrovipera lebetina snake venom and investigated its anti-tumoral effect on its own or combined with cisplatin, in human melanoma cells. Methods Biochemical characterization, in vitro cells assays such as viability, apoptosis, adhesion, migration, invasion, Western blotting and in silico analysis were used in this study. Results Macrovipecetin decreased melanoma cell viability 100 times more than cisplatin. Interestingly, when combined with the drug, macrovipecetin enhanced the sensitivity of SK-MEL-28 cells by augmenting their apoptosis through increased expression of the apoptosis inducing factor (AIF) and activation of ERK1/2, p38, AKT and NF-κB. Moreover, macrovipecetin alone or combined with cisplatin induced the expression of TRADD, p53, Bax, Bim and Bad and down-regulated the Bcl-2 expression and ROS levels in SK-MEL-28 cells. Interestingly, these treatments impaired SK-MEL-28 cell adhesion, migration and invasion through modulating the function and expression of αvβ3 integrin along with regulating E-cadherin, vimentin, β-catenin, c-Src and RhoA expression. In silico study suggested that only the α chain of macrovipecetin interacts with a region overlapping the RGD motif binding site on this integrin. Conclusions We validated the antitumor effect of macrovipecetin when combined, or not, with cisplatin on SK-MEL-28 cells. General significance The presented work proposes the potential use of macrovipecetin and cisplatin in combination as an effective anti-melanoma treatment.

Background Combination medicines including an artemisinin are the mainstay of antimalarial therapy. Artemisinins are potent embryotoxicants in animal species due to their trioxane moiety. Methods As part of its development, the new synthetic trioxolane antimalarial artefenomel (OZ439) was tested in rat whole embryo culture and in rat embryo-fetal toxicity studies with dosing throughout organogenesis or with a single dose on Gestational Day (GD) 12. The single-dose studies included groups treated with artesunate to allow a direct comparison of the embryotoxicity of the two antimalarials and included toxicokinetics hematology and histological examination of embryos. In addition, the distribution of artefenomel-related material in plasma was determined after the administration of 14 C-artefenomel. Results Artefenomel and artesunate showed similar patterns of embryotoxicity including cardiovascular defects and resorption with a steep dose-response. They both also caused a depletion of circulating embryonic erythroblasts both in vitro and in vivo and decreases in maternal reticulocyte count. However, artefenomel was ∼250-fold less potent than the active metabolite of artesunate (dihydroartemisinin) as an embryotoxicant in vitro. The safety margin (based on AUC) for artefenomel administered on GD 12 was approximately 100-fold greater than that for artesunate. Also, unlike artesunate, artefenomel was not a selective developmental toxicant. Conclusions The lesser embryotoxicity of artefenomel is likely linked to its original design which included two blocking side groups that had been introduced to lower the reactivity with ferrous iron. Our data support the hypothesis that artefenomel's improved safety margin is linked to a lower potential for inhibiting heme biosynthesis in embryonic erythroblasts.

Background\ud \ud The impact of temporal feeding patterns remains a major unanswered question in nutritional science. Progress has been hampered by the absence of a reliable method to impose temporal feeding in laboratory rodents, without the confounding influence of food-hoarding behavior.\ud Objective\ud \ud The aim of this study was to develop and validate a reliable method for supplying crushed diets to laboratory rodents in consistent, relevant feeding patterns for prolonged periods.\ud Methods\ud \ud We programmed our experimental feeding station to deliver a standard diet [StD; Atwater Fuel Energy (AFE) 13.9% fat] or high-fat diet (HFD; AFE 45% fat) during nocturnal grazing [providing 1/24th of the total daily food intake (tdF/I) of ad libitum–fed controls every 30 min] and meal-fed (3 × 1-h periods of ad libitum feeding) patterns in male rats (Sprague-Dawley: 4 wk old, 72–119 g) and mice [C57/Bl6J wild-type (WT): 6 mo old, 29–37 g], and ghrelin-null littermates (Ghr−/−; 27–34 g).\ud Results\ud \ud Grazing yielded accurate, consistent feeding events in rats, with an approximately linear rise in nocturnal cumulative food intake [tdF/I (StD): 97.4 ± 1.5% accurate compared with manual measurement; R2 = 0.86; tdF/I (HFD): 99.0 ± 1.4% accurate; R2 = 0.86]. Meal-feeding produced 3 nocturnal meals of equal size and duration in StD-fed rats (tdF/I: 97.4 ± 0.9% accurate; R2 = 0.90), whereas the second meal size increased progressively in HFD-fed rats (44% higher on day 35 than on day 14; P < 0.01). Importantly, cumulative food intake in grazing and meal-fed rats was identical. Similar results were obtained in WT mice except that less restricted grazing induced hyperphagia (compared with meal-fed WT mice; P < 0.05 from day 1). This difference was abolished in Ghr−/− mice, with meal initiation delayed and meal duration enhanced. Neither pattern elevated corticosterone secretion in rats, but meal-feeding aligned ultradian pulses.\ud Conclusions\ud \ud We have established a consistent, measurable, researcher-defined, stress-free method for imposing temporal feeding patterns in rats and mice. This approach will facilitate progress in understanding the physiologic impact of feeding patterns.

A phenotypic screen of a diverse library of small molecules for inhibition of the development of larvae of the parasitic nematode Haemonchus contortus led to the identification of a 1-methyl-1H-pyrazole-5-carboxamide derivative with an IC50 of 0.29 μM. Medicinal chemistry optimization targeted modifications on the left-hand side (LHS), middle section, and right-hand side (RHS) of the scaffold in order to elucidate the structure–activity relationship (SAR). Strong SAR allowed for the iterative and directed assembly of a focus set of 64 analogues, from which compound 60 was identified as the most potent compound, inhibiting the development of the fourth larval (L4) stage with an IC50 of 0.01 μM. In contrast, only 18% inhibition of the mammary epithelial cell line MCF10A viability was observed, even at concentrations as high as 50 μM.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts., Author Summary Malaria leads to the loss of over 440,000 lives annually; accelerating research to discover new candidate drugs is a priority. Medicines for Malaria Venture (MMV) has distilled over 25,000 compounds that kill malaria parasites in vitro into a group of 400 representative compounds, called the "Malaria Box". These Malaria Box sets were distributed free-of-charge to research laboratories in 30 different countries that work on a wide variety of pathogens. Fifty-five groups compiled >290 assay results for this paper describing the many activities of the Malaria Box compounds. The collective results suggest a potential mechanism of action for over 130 compounds against malaria and illuminate the most promising compounds for further malaria drug development research. Excitingly some of these compounds also showed outstanding activity against other disease agents including fungi, bacteria, other single-cellular parasites, worms, and even human cancer cells. The results have ignited over 30 drug development programs for a variety of diseases. This open access effort was so successful that MMV has begun to distribute another set of compounds with initial activity against a wider range of infectious agents that are of public health concern, called the Pathogen Box, available now to scientific labs all over the world (www.PathogenBox.org).

The relationship between the degree of GH deficiency and impaired bone integrity is not simple and may be influenced by related endocrine variables. To test the hypothesis that elevated adiposity and hyperleptinaemia are contributory factors, we quantified femoral trabecular organisation in two models of GH deficiency with divergent degrees of adiposity - the moderately GH-deficient/hyperleptinaemic transgenic growth retarded (Tgr) rat and the profoundly GH-deficient/hypoleptinaemic dw/dw rat. Trabecular density (bone volume/total volume) and surface were reduced by 16% in dw/dw males, with a more fragmented trabecular lattice. This impairment was more pronounced in Tgr rats, with trabecular number and density further reduced (by an additional 21%) and relative surface (bone surface/bone volume), trabecular convexity (structural modal index) and fragmentation (pattern factor) increased. To establish whether the presence of obesity/hyperleptinaemia exacerbates bone impairment in GH deficiency, trabecular structure was assessed in dw/dw rats following diet-induced obesity (DIO). DIO had minimal effect on trabecular architecture, the increased concavity of trabecular surfaces being the only observable effect. Similarly, infusion of leptin into the tibial bone marrow cavity had no effect on trabecular organisation or tibial growth in wild-type rats. However, while this procedure also failed to affect trabecular architecture or osteoclast number in dw/dw rats, distal osteoblast surface was increased by 23%, marrow adipocyte number and epiphyseal plate width being reduced (by 40 and 5% respectively), without increasing caspase-3 immunoreactivity. These findings suggest that while leptin may directly inhibit adipocyte differentiation and favour osteoblast production, hyperleptinaemia makes only a minimal contribution to the impairment of bone structure in GH deficiency.

Growth hormone (GH)-deficiency is usually associated with elevated adiposity, hyperleptinemia, and increased fracture risk. Since leptin is thought to enhance cortical bone formation, we have investigated the contribution of elevated adiposity and hyperleptinemia on femoral strength in rodent models of GH deficiency. Quantification of the transpubertal development of femoral strength in the moderately GH-deficient/hyperleptinemic Tgr rat and the profoundly GH-deficient/hypoleptinemic dw/dw rat revealed that the mechanical properties of cortical bone in these two models were similarly compromised, a 25–30% reduction in failure load being entirely due to impairment of geometric variables. In contrast, murine models of partial (GH antagonist transgenic) and complete (GH receptor-null) loss of GH signaling and elevated adiposity showed an impairment of femoral cortical strength proportionate to the reduction of GH signaling. To determine whether impaired femoral strength is exacerbated by obesity/hyperleptinemia, femoral strength was assessed in dw/dw rats following two developmental manipulations that elevate abdominal adiposity and circulating leptin, neonatal monosodium glutamate (MSG) treatment, and maintenance on an elevated fat diet. The additional impairment of femoral strength following MSG treatment is likely to have resulted from a reduction in residual activity of the hypothalamo-pituitary-GH-IGF-I axis, but consumption of elevated dietary fat, which did not reduce circulating IGF-I, failed to exacerbate the compromised femoral strength in dw/dw rats. Taken together, our data indicate that the obesity and hyperleptinemia usually associated with GH deficiency do not exert a significant influence over the strength of cortical bone.

Development of a safe, effective, and inexpensive therapy for African trypanosomiasis is an urgent priority. In this study, we evaluated the validity of Trypanosoma brucei glycogen synthase kinase 3 (GSK-3) as a potential drug target. Interference with the RNA of either of two GSK-3 homologues in bloodstream-form T. brucei parasites led to growth arrest and altered parasite morphology, demonstrating their requirement for cell survival. Since the growth arrest after RNA interference appeared to be more profound for T. brucei GSK-3 “short” (Tb10.161.3140) than for T. brucei GSK-3 “long” (Tb927.7.2420), we focused on T. brucei GSK-3 short for further studies. T. brucei GSK-3 short with an N-terminal maltose-binding protein fusion was cloned, expressed, and purified in a functional form. The potency of a GSK-3-focused inhibitor library against the recombinant enzyme of T. brucei GSK-3 short, as well as bloodstream-form parasites, was evaluated with the aim of determining if compounds that inhibit enzyme activity could also block the parasites' growth and proliferation. Among the compounds active against the cell, there was an excellent correlation between activity inhibiting the T. brucei GSK-3 short enzyme and the inhibition of T. brucei growth. Thus, there is reasonable genetic and chemical validation of GSK-3 short as a drug target for T. brucei . Finally, selective inhibition may be required for therapy targeting the GSK-3 enzyme, and a molecular model of the T. brucei GSK-3 short enzyme suggests that compounds that selectively inhibit T. brucei GSK-3 short over the human GSK-3 enzymes can be found.

SummaryTyrosine kinases and phosphatases establish the crucial balance of tyrosine phosphorylation in cellular signaling, but creating specific inhibitors of protein Tyr phosphatases (PTPs) remains a challenge. Here, we report the development of a potent, selective inhibitor of Mycobacterium tuberculosis PtpB, a bacterial PTP that is secreted into host cells where it disrupts unidentified signaling pathways. The inhibitor, (oxalylamino-methylene)-thiophene sulfonamide (OMTS), showed an IC50 of 440 ± 50 nM and >60-fold specificity for PtpB over six human PTPs. The 2 Å resolution crystal structure of PtpB in complex with OMTS revealed a large rearrangement of the enzyme, with some residues shifting >27 Å relative to the PtpB:PO4 complex. Extensive contacts with the catalytic loop provide a potential basis for inhibitor selectivity. Two OMTS molecules bound adjacent to each other, raising the possibility of a second substrate phosphotyrosine binding site in PtpB. The PtpB:OMTS structure provides an unanticipated framework to guide inhibitor improvement.

The biological activity of chemokines requires interactions with cell surface proteoglycans. We have determined the structure of the chemokine RANTES (regulated on activation normal T cell expressed) in the presence of heparin-derived disaccharide analogs by X-ray crystallography. These structures confirm the essential role of the BBXB motif in the interaction between the chemokine and the disaccharide. Unexpected interactions were observed in the 30s loop and at the amino terminus. Mutant RANTES molecules were designed to abrogate these interactions and their biological activity examined in vivo. The K45E mutant within the BBXB motif lost the capacity to bind heparin and the ability to elicit cellular recruitment. The Y3A mutant maintained its capacity to bind heparin but was unable to elicit cellular recruitment. Finally, a tetrasaccharide is the smallest oligosaccharide which effectively abolishes the ability of RANTES to recruit cells in vivo. These crystallographic structures provide a description of the molecular interaction of a chemokine with glycosaminoglycans.

Binding of interleukin-8 (IL-8) to glycosaminoglycans (GAGs) on the surface of endothelial cells is crucial for the recruitment of neutrophils to an inflammatory site. Deriving structural knowledge about this interaction from in silico docking experiments has proved difficult because of the high flexibility and the size of GAGs. Therefore, we developed a docking method that takes into account ligand and protein flexibility by running approximately 15,000 molecular dynamics simulations of the docking event with different initial orientations of the binding partners. The method was shown to successfully reproduce the residues of basic fibroblast growth factor involved in GAG binding. Docking of a heparin hexasaccharide to IL-8 gave an interaction interface involving the basic residues His18, Lys20, Arg60, Lys64, Lys67, and Arg68. By subjecting IL-8 single-site mutants, in which these amino acids were replaced by alanine, to isothermal fluorescence titrations, the affinities for heparin were determined to be wtIL-8 > IL-8(H18A) >> IL-8(R68A) > IL-8(K67A) >> IL-8(K20A) > IL-8(R60A) >> IL-8(K64A). A comparison with the binding energies calculated from the model revealed high values for wtIL-8 and the H18A mutant and significantly lower but similar energies for the remaining mutants. Connecting the two fully sulfated hexasaccharides bound to each of the two IL-8 monomers in the dimeric chemokine by an N-acetylated dodecasaccharide gave a complex structure in which the GAG molecule aligned in a parallel fashion to the N-terminal alpha-helices of IL-8 like a horseshoe. A 5-ns molecular dynamics simulation of this complex confirmed its structural stability and revealed a reorientation in both binding sites where a disaccharide became the central binding unit. Isothermal fluorescence titration experiments using differently sulfated heparin disaccharides confirmed that a single disaccharide can indeed bind IL-8 with high affinity.

The understanding of the mechanisms underlying eosinophil recruitment in vivo may aid in the development of novel strategies for the treatment of allergic disorders. In this study, we investigated the role of chemokines in the cascade of events leading to eosinophil recruitment in a stem cell factor (SCF)- and leukotriene B4 (LTB4)-dependent allergic pleurisy model in mice. The intrapleural administration of the eosinophil-active chemokines eotaxin, RANTES, and macrophage-inflammatory protein 1α (MIP-1α) induced a time- and dose-dependent eosinophil recruitment. Pretreatment with anti-eotaxin, but not anti-RANTES or anti-MIP-1α, blocked the recruitment of eosinophils following Ag challenge of sensitized animals, and significant eotaxin immunoreactivity was detected in the pleural cavity of these animals. Similarly, only the anti-eotaxin inhibited the eosinophil recruitment induced by injection of SCF in naive animals. However, blockade of SCF did not inhibit the release of eotaxin after Ag challenge of sensitized mice. Akin to its effects on SCF and in the allergic reaction, eotaxin-induced eosinophil recruitment was blocked by the LTB4 receptor antagonist CP105696. Nevertheless, SCF, but not eotaxin, appeared to regulate the endogenous release of LTB4 after Ag challenge. Finally, we show that low doses of eotaxin synergized with LTB4 to induce eosinophil recruitment in the pleural cavity. Overall, the present results show that eotaxin and SCF-induced LTB4 cooperate to induce eosinophil recruitment into sites of allergic inflammation. Cooperation between inflammatory mediators must be an important phenomenon in vivo, explaining both the ability of lower concentrations of mediators to induce a full-blown functional response and the effectiveness of different strategies at inhibiting these responses.

The transgenic growth retarded (Tgr) rat is the first genetic model of growth hormone (GH) deficiency whose growth can be accelerated with exogenous GH secretagogues (GHSs). In this study, we have demonstrated that GHS-receptor (GHS-R) mRNA expression in the arcuate nucleus of Tgr rats was not significantly different to that in wild-type littermates. We have confirmed that GHS-induced elevation in body weight gain was accompanied by acceleration of skeletal growth, and that the effects of the GHS, GHRP-6, were both dose- and pattern-dependent. The growth response with continuous infusion of GHRP-6 was transient, accompanied by suppression of GH and corticosterone responses to bolus injection of GHRP-6. This desensitization occurred without downregulation of arcuate GHS-R mRNA expression, but was accompanied by elevated periventricular somatostatin mRNA expression. In contrast, pulsatile (3-hourly) infusion of GHRP-6 produced sustained growth and GH responses, which were accompanied by suppression of corticosterone responses and elevated arcuate GH-releasing factor (GRF) mRNA expression. Skeletal growth was further accelerated by coinfusion of GRF, but significant depletion of pituitary GH stores suggested that this growth rate may not be sustainable. These experiments confirm the importance of the Tgr rat for investigating the growth promoting potential of the GHSs in the context of GH-deficient dwarfism, and suggest that elevated somatostatin expression may mediate the suppression of the GRF-GH and hypothalamo-pituitary-adrenal axes following continuous GHRP-6 treatment.

The chemokine RANTES (regulated on activation normal T cell expressed and secreted; CCL5) binds selectively to glycosaminoglycans (GAGs) such as heparin, chondroitin sulfate, and dermatan sulfate. The primary sequence of RANTES contains two clusters of basic residues, (44)RKNR(47) and (55)KKWVR(59). The first is a BBXB motif common in heparin-binding proteins, and the second is located in the loop directly preceding the C-terminal helix. We have mutated these residues to alanine, both as point mutations as well as triple mutations of the 40s and 50s clusters. Using a binding assay to heparin beads with radiolabeled proteins, the (44)AANA(47) mutant demonstrated an 80% reduction in its capacity to bind heparin, whereas the (55)AAWVA(59) mutant retained full binding capacity. Mutation of the (44)RKNR(47) site reduced the selectivity of RANTES binding to different GAGs. The mutants were tested for their integrity by receptor binding assays on CCR1 and CCR5 as well as their ability to induce chemotaxis in vitro. In all assays the single point mutations and the triple 50s cluster mutation caused no significant difference in activity compared with the wild type sequence. However, the triple 40s mutant showed a 80-fold reduction in affinity for CCR1, despite normal binding to CCR5. It was only able to induce monocyte chemotaxis at micromolar concentrations. The triple 40s mutant was also able to inhibit HIV-1 infectivity, but consistent with its abrogated GAG binding capacity, it no longer induced enhanced infectivity at high concentrations.

Platelets are known to contain platelet factor 4 and β-thromboglobulin, α-chemokines containing the CXC motif, but recent studies extended the range to the β-family characterized by the CC motif, including RANTES and Gro-α. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors. Polymerase chain reaction detected chemokine receptor messenger RNA in platelet RNA. CCR1, CCR3, and especially CCR4 gave strong signals; CXCR1 and CXCR4 were weakly positive. Flow cytometry with specific antibodies showed the presence of a clear signal for CXCR4 and weak signals for CCR1 and CCR3, whereas CXCR1, CXCR2, CXCR3, and CCR5 were all negative. Immunoprecipitation and Western blotting with polyclonal antibodies to cytoplasmic peptides clearly showed the presence of CCR1 and CCR4 in platelets in amounts comparable to monocytes and CCR4 transfected cells, respectively. Chemokines specific for these receptors, including monocyte chemotactic protein 1, macrophage inflammatory peptide 1α, eotaxin, RANTES, TARC, macrophage-derived chemokine, and stromal cell–derived factor 1, activate platelets to give Ca++ signals, aggregation, and release of granule contents. Platelet aggregation was dependent on release of adenosine diphosphate (ADP) and its interaction with platelet ADP receptors. Part, but not all, of the Ca++ signal was due to ADP release feeding back to its receptors. Platelet activation also involved heparan or chondroitin sulfate associated with the platelet surface and was inhibited by cleavage of these glycosaminoglycans or by heparin or low molecular weight heparin. These platelet receptors may be involved in inflammatory or allergic responses or in platelet activation in human immunodeficiency virus infection.

Chemokines are 8-10 kDa proteins involved in the control of leukocyte trafficking and activation. In free solution, chemokines are monomers at physiologic concentrations, although many multimerize at higher concentrations. Cell surface heparan sulfate may sequester chemokines, increasing their local concentrations and facilitating their binding to receptors expressed on leukocytes. In competitive binding assays using immobilized heparin, a 2-3-fold increase in the bound radiolabeled chemokine was seen with increasing concentrations of unlabeled chemokine in the nanomolar range. Unlabeled chemokine concentrations between 0.25 and 50 microM were needed to compete the bound radioactivity. This biphasic competition curve was not seen for N-methyl-L25 IL-8, a variant of IL-8 which is unable to dimerize. In addition, complexes of chemokine and heparin eluted from gel filtration columns with apparent molecular masses of 33-60 kDa, suggesting that chemokine multimerization had occurred. The physiological relevance of this multimerization process was seen from studies using human endothelial cells. The endothelial cell binding sites for IL-8, RANTES, and MCP-1 were deduced to be glycosaminoglycans since competition assays showed the biphasic curves and micromolar IC50 values seen in studies with immobilized heparin, and mRNA for known chemokine receptors was not detected. Furthermore, digestion of endothelial cell monolayers with glycosaminidases decreased chemokine binding by up to 80%. Glycosaminoglycans can act as modulators of the ligand binding affinity of chemokine receptor-bearing cells. Removal of glycosaminoglycans from CHO cells expressing chemokine receptors CXCR1, CCR1, or CCR2 resulted in 40-70% decreases in the binding of RANTES, MCP-1, IL-8, and MIP-1alpha. Our data show that cell surface glycosaminoglycans induce polymerization of chemokines, increasing their local concentration and therefore enhancing their effects on high-affinity receptors within the local microenvironment.

Candida albicans phosphomannose isomerase (PMI) is a zinc metalloprotein of known crystal structure. When heterologously overexpressed in Escherichia coli, a blue protein that contains up to 0.5 iron atoms/PMI molecule could be isolated, with absorption maxima at 420 nm and 680 nm. These bands are reminiscent of ferric catecholate complexes, an assignment that has been confirmed by resonance Raman spectroscopy, and by reaction with Arnow's reagent, which is specific for the presence of 3,4-dihydroxy-phenylalanine (Dopa). After enzymatic digestion of blue PMI, a peptide with the sequence DPHAXISG was isolated corresponding to residues Asp283 -Gly290 in the amino acid sequence of C. albicans PMI, where the unidentified residue X287 is encoded by a tyrosine codon. It is proposed that iron and oxygen bring about hydroxylation of Tyr287 in PMI and that Fe(III) subsequently chelates the Dopa residue to give the characteristic absorption spectrum. The EPR spectrum of the blue protein suggests three iron environments in the protein, two in axial environments with E/D values approximately equal to 0.06 and 0.12 and one rhombic species. The nature of the iron co-ordination sites is discussed with the help of model systems and by comparison with other blue non-heme iron proteins.

Connective tissue-activating peptide III (CTAP-III) and neutrophil-activating peptide-2 (NAP-2) are both derived from a common precursor, platelet basic protein (PBP), which is stored in the alpha-granules of platelets and released upon their activation. CTAP-III is an 85-residue peptide which is converted to NAP-2 by enzymic removal of the 15 amino-terminal residues. Both peptides play a role in the early stages of wound healing and inflammation through different activities. We have cloned the cDNA for PBP and expressed constructs coding for the CTAP-III and NAP-2 polypeptides in Escherichia coli. We have purified and renatured these recombinant proteins. The integrity of the recombinant proteins has been ascertained by in vitro bioassays. CTAP-III causes 51% histamine release from the basophilic cell lin KU812 at 10(-7) M, whereas NAP-2 only causes 28% release at the same concentration. In assays on human neutrophils, NAP-2 had an EC50 of 2 x 10(-8) M in chemotaxis, an EC50 of 3 x 10(-8) M for shape change, and could displace IL-8 from neutrophils with a Kd of 7.5 x 10(-9) M. CTAP-III had no activity in these assays. The disulfide bonds have been identified by peptide mapping and sequence analysis, and are in the positions predicted by homology to interleukin-8 and platelet factor 4. Measurement of the molecular mass at physiologic concentrations by gel permeation chromatography has shown that CTAP-III forms predominantly tetramers and dimers, whereas NAP-2 is only dimetric. SDS/PAGE analysis of samples cross-linked with disuccinimidyl suberate support these topologies. We postulate a mechanism for tetramer formation based on the interaction of the amino-terminal extension in CTAP-III involving a helix-helix interaction that could stabilize the association of two CTAP-III dimers.

We have tested five distinct classes of established and experimental antimalarial drugs for their anticancer potential, using a panel of 91 human cancer lines. Three classes of drugs: artemisinins, synthetic peroxides and DHFR (dihydrofolate reductase) inhibitors effected potent inhibition of proliferation with IC 50 s in the nM- low µM range, whereas a DHODH (dihydroorotate dehydrogenase) and a putative kinase inhibitor displayed no activity. Furthermore, significant synergies were identified with erlotinib, imatinib, cisplatin, dasatinib and vincristine. Cluster analysis of the antimalarials based on their differential inhibition of the various cancer lines clearly segregated the synthetic peroxides OZ277 and OZ439 from the artemisinin cluster that included artesunate, dihydroartemisinin and artemisone, and from the DHFR inhibitors pyrimethamine and P218 (a parasite DHFR inhibitor), emphasizing their shared mode of action. In order to further understand the basis of the selectivity of these compounds against different cancers, microarray-based gene expression data for 85 of the used cell lines were generated. For each compound, distinct sets of genes were identified whose expression significantly correlated with compound sensitivity. Several of the antimalarials tested in this study have well-established and excellent safety profiles with a plasma exposure, when conservatively used in malaria, that is well above the IC 50 s that we identified in this study. Given their unique mode of action and potential for unique synergies with established anticancer drugs, our results provide a strong basis to further explore the potential application of these compounds in cancer in pre-clinical or and clinical settings.

Several members of the chemokine receptor family have been shown to function in association with CD4 to permit HIV-1 entry and infection. However, the mechanism by which these molecules serve as CD4-associated cofactors is unclear. In the present report, we show that one member of this family, termed Fusin/CXCR4, is able to function as an alternative receptor for some isolates of HIV-2 in the absence of CD4. This conclusion is supported by the finding that (1) CD4-independent infection by these viruses is inhibited by an anti-Fusin monoclonal antibody, (2) Fusin expression renders human and nonhuman CD4-negative cell lines sensitive to HIV-2-induced syncytium induction and/or infection, and (3) Fusin is selectively down-regulated from the cell surface following HIV-2 infection. The finding that one chemokine receptor can function as a primary viral receptor strongly suggests that the HIV envelope glycoprotein contains a binding site for these proteins and that differences in the affinity and/or the availability of this site can extend the host range of these viruses to include a number of CD4-negative cell types.

Phosphomannose isomerase (PMI) catalyses the interconversion of mannose 6-phosphate and fructose 6-phosphate in prokaryotic and eukaryotic cells. The enzyme is a metalloenzyme which contains 1 mol of zinc per mol of enzyme. Heterologous expression of the cDNA coding for the Candida albicans enzyme in the prokaryotic host Escherichia coli results in an expression level of up to 30% of total E. coli protein. Ten percent of recombinant PMI is expressed in the soluble fraction and 90% in inclusion bodies. Inclusion of a high level of zinc in the fermentation medium resulted in a fourfold increase in soluble protein. Co-expression of the bacterial chaperones, GroES and GroEL, resulted in a proportional twofold increase in soluble PMI while causing an overall decrease in the PMI expression level. Folding denatured PMI in vitro required reductant and zinc ions. The yield of renatured protein was increased by folding in the presence of GroEL and DnaK in an ATP-independent manner. The refolding yield of denatured soluble enzyme from a guanidine solution was threefold higher than that of folding monomerized inclusion body protein solubilized in guanidine hydrochloride. This suggests that a proportion of recombinant protein expressed in E. coli inclusion bodies may be irreversibly denatured.

Phosphomannose isomerase (PMI) catalyses the reversible isomerization of fructose-6-phosphate (F6P) and mannose-6-phosphate (M6P). Absence of PMI activity in yeasts causes cell lysis and thus the enzyme is a potential target for inhibition and may be a route to antifungal drugs. The 1.7 ˚ crystal structure of PMI from Candida albicans shows that the enzyme has three distinct domains. The active site lies in the central domain, contains a single essential zinc atom, and forms a deep, open cavity of suitable dimensions to contain M6P or F6P. The central domain is flanked by a helical domain on one side and a jelly-roll like domain on the other.

Interleukin-8 (IL-8), a member of the CXC chemokine family, is a key activator of neutrophils. We have previously shown that two novel CC chemokine-like properties, namely monocyte chemoattraction and binding to CC CKR-1, are introduced into IL-8 by mutating Leu25 to the conserved tyrosine present in CC chemokines. To further investigate the role of this position in receptor selectivity, we have mutated Leu25 to cysteine. The protein folds correctly with two disulfide bonds and a free thiol group at Cys25. This mutant behaves overall like wild-type IL-8, with little change in neutrophil chemotaxis and IL-8 receptor binding, and has no effect on CC CKR-1. These data are consistent with cysteine being approximately isosteric with the natural amino acid leucine. However, modification of the cysteine by addition of a fluorescent N-methyl-N-(2-N-methyl, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminoethyl)acetamido (NBD) group lowers potency in neutrophil chemotaxis and affinity in IL-8 receptor binding assays by 2 orders of magnitude. This Leu25xrarr; Cys-NBD mutant introduces monocyte chemoattractant activity and the ability to displace 125I-labeled macrophage inflammatory protein-1α from the recombinant CC CKR-1 receptor. Additionally, we show a specific interaction between the fluorescent mutant and the N-terminal 34-amino acid peptide from CC CKR-1. This confirms the importance of this region in IL-8 in receptor binding and in conferring specificity between CXC and CC chemokines. Circular dichroism spectra of the IL-8 mutants having CC chemokine-like activity show a consistent drop in α-helical content compared with the spectra for wild-type IL-8. This suggests that distortion of the C-terminal helix may play a role in chemokine receptor-ligand selectivity.

We have cloned the murine homologue of human CC Chemokine Receptor-4 (CC CKR-4). In equilibrium competition binding assays performed in undifferentiated HL-60 cells transfected with human and murine CC CKR-4 cDNA, the IC 50 values for the binding of [ 125 I]macrophage inflammatory protein-1α to human and murine CC CKR-4 were 14.5 ± 9.0 nM and 10.1 ± 3.0 nM, respectively, and the IC 50 values for the binding of [ 125 I]RANTES to human and murine CC CKR-4 were 9.3 ± 3.0 nM and 5.7 ± 2.6 nM, respectively. The cDNA clone for murine CC CKR-4 is 1531 bp, and the largest open reading frame encodes a protein of 360 amino acids that is 85% identical to human CC CKR-4. Murine CC CKR-4 was detected in the thymus and T-cell lines by Northern blot analysis. This first report of direct binding of chemokines to CC CKR-4 demonstrates that the highly homologous human and murine receptors have similar binding characteristics and tissue distribution.

The chemokine superfamily can be sub-divided into two groups based on their amino terminal cysteine spacing. The CXC chemokines are primarily involved in neutrophil-mediated inflammation and, so far, two human receptors have been cloned. The CC chemokines tend to be involved in chronic inflammation, and recently we have cloned a fourth leukocyte receptor for this group of ligands. Understanding what makes one receptor bind its range of agonists is important if we are to develop potent selective antagonists. We have started to investigate the molecular basis of this receptor selectivity by looking at why CC chemokines do not bind to the CXC receptors in several ways. First, we looked at the role of the three-dimensional structure of the ligand, and have solved the three-dimensional structure of RANTES using nuclear magnetic resonance spectroscopy. The structure is similar to that already determined for the CC chemokine macrophage inflammatory protein-1β, and it has a completely different dimer interface to that of the CXC chemokine interleukin-8 (IL-8). However, the monomer structures of all the chemokines are very similar, and at physiological concentrations the proteins are likely to be monomeric. Second, by examining all the known CC and CXC chemokines, we have found a region that differs between the two subfamilies. Mutations of one of the residues in this region, Leu-25 in IL-8, to tyrosine (which is conserved at this position in CC chemokines) enables the mutant IL-8 to bind CC-chemokine receptor-1 (CC-CKR-1) and introduces monocyte chemoattractant activity. Using other mutations in this region, we can show a direct interaction with the N-terminus of CC-CKR-1. Third, we have found that modification of the amino terminus of RANTES by addition of one amino acid makes it into an antagonist with nanomolar potency. Taken together, this data suggests a two-site model for receptor activation and for selectivity between CC and CXC chemokines, with an initial receptor contact provided by the main body of the chemokine, and activation provided by the amino terminal region.

The CC chemokines RANTES and MIP-1 alpha are known to activate certain leucocytes and leucocytic cell lines. We have produced and fully characterised the recombinant proteins expressed in E. coli. They induce chemotaxis of the pro-monocytic cell line, THP-1 and T cells. THP-1 cells express three of the known CC chemokine receptors. In order to study the activation of a single receptor, we have expressed the shared receptor (CC CKR-1) for RANTES and MIP-1 alpha stably in the HEK 293 cell line. We have examined the effects of RANTES and MIP-1 alpha on the CC CKR-1 transfectants by equilibrium binding studies and in a chemotaxis assay. RANTES competes for [125I]RANTES with an IC50 of 0.6 +/- 0.23 nM, whereas MIP-1 alpha competes for its radiolabelled counterpart with an IC50 of 10 +/- 1.6 nM in the transfectants. These affinities are the same as those measured on the THP-1 cell line. The stably transfected HEK 293 cells respond to both these chemokines in the chemotaxis assay with the same EC50 values as those measured for THP-1 cells. This indicates that this cellular response can be mediated through the CC CKR-1 receptor.

In order to study the role of platelets in inflammation we constructed a cDNA library from human platelet mRNA. By polymerase chain reaction (PCR) analysis of the library we have shown that platelets express mRNAs for the following chemokines: connective tissue activating peptide-III(CTAP-III), epithelial-derived neutrophil activating factor-78 (ENA-78), RANTES and monocyte chemotactic protein-3 (MCP-3). Platelets also express mRNAs for interleukin 8 receptor A (IL-8RA) and a novel chemokine receptor K5.5. These results suggest that chemokines may not only play an important role in platelet activation but can also influence the nature of the leukocyte infiltrate to sites of inflammation and infection, by the production of multiple chemokines with overlapping specificities.

We report the cloning and characterization of a novel basophil CC chemokin receptor, K5-5, from the human immature basophilic cell line KU-812. The predicted protein sequence of K5-5 shows only 49% identity to the macrophage inflammatory protein-1 alpha/RANTES receptor (CC CKR-1) and 47% identity to monocyte chemotactic protein-1 receptor (b form), suggesting that this cDNA encodes a novel member of the CC chemokine receptor family. Analysis of K5-5 mRNA expression indicates that it is restricted to leukocyte-rich tissues. In addition, we have shown significant levels of K5-5 mRNA in human basophils, which were up-regulated by treatment with interleukin-5. The CC chemokines, Macrophage inflammatory protein-1 alpha, RANTES, and monocyte chemotactic protein-1 were able to stimulate a Ca(2+)-activated chloride channel in Xenopus laevis oocytes injected with K5-5 cRNA, whereas no signal was detected in response to monocyte chemotactic protein-2, macrophage inflammatory protein-1 beta, or the CXC chemokine, interleukin-8. Taken together, these results indicate for the first time the presence of a CC chemokine receptor on basophils, which functions as a "shared" CC chemokine receptor and may therefore be implicated in the pathogenesis of basophil-mediated allergic diseases.

The solution structure of the chemokine RANTES (regulated on activation, normal T-cell expressed and secreted) has been determined using NMR spectroscopy. Backbone and side-chain 1H and 15N assignments have been obtained using a combination of two-dimensional homonuclear and three-dimensional heteronuclear spectra. Regular elements of secondary structure have been identified on the basis of a qualitative interpretation of NOE data, J(NH-H alpha) coupling constants, and amide exchange rates. Three-dimensional structures were calculated from a total of 2146 experimental restraints using a combination of distance geometry and simulated annealing protocols. For the 13 best structures the average backbone (N, C alpha, C) atomic rmsd from the mean coordinates for residues 5-65 is 0.64 A (+/- 0.14 A) for the dimer and 0.50 A (+/- 0.08 A) for the individual monomers. Each monomer consists of a three-stranded antiparallel beta-sheet (residues 26-30, 38-43, 48-51) in a Greek key motif with a C-terminal helix (56-65) packed across the sheet, an arrangement similar to the monomeric structure of other members of this chemokine family (IL-8, PF4, MGSA/Gro alpha, and MIP-1 beta). Overall, the RANTES dimer resembles that previously reported for MIP-1 beta.

Silver ions and silver-containing compounds have been used as topical antimicrobial agents in a variety of clinical situations. We have previously shown that the enzyme phosphomannose isomerase (PMI) is essential for the biosynthesis of Candida albicans cell walls. In this study, we find that PMI can be inhibited by silver ions. This process is shown to be irreversible, and is a two-step process, involving an intermediate complex with a dissociation constant, Ki, of 59 +/- 8 microM, and a maximum rate of inactivation of 0.25 +/- 0.04 min-1 in 50 mM Hepes buffer, pH 8.0 at 37 degrees C. The enzyme can be protected against this inactivation by the substrate mannose 6-phosphate, with a dissociation constant of 0.31 +/- 0.04 mM, close to its Km value. Flamazine (silver sulfadiazine) is a silver-containing antibiotic which is used clinically as a topical antimicrobial and antifungal agent. We compared the ability of silver sulfadiazine and two other silver-containing compounds to irreversibly inactivate C. albicans PMI. The addition of the organic moiety increased the affinity of the compounds, with silver sulfadiazine showing a Ki of 190 +/- 30 nM. In all cases, the maximum inhibition rate was similar, implying a similar rate-determining step. Silver sulfadiazine does not inhibit Escherichia coli PMI, and this suggests a role of the only free cysteine, Cys-150, in the inactivation process. To confirm this, we mutated this residue to alanine in C. albicans PMI. The resultant Cys150 --> Ala mutant protein showed similar Vm and Km values to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

We have purified platelet factor 4 from porcine platelets and shown that it is glycosylated. The purified protein migrated as a broad band at approximately 14,000 Da, characteristic of glycoproteins. Electrospray mass spectroscopy of the intact protein gave a predominant mass of 11,111 Da, with a minor component of 10,804 Da. Sialidase digestion reduces both forms to a single mass of 10,497 Da. Upon Edman degradation, the amino terminus was found to be blocked by the presence of a pyroglutamate residue. We have determined the complete primary structure of platelet factor 4 by peptide mapping and Edman degradation, thereby completing information on the amino-terminal and carboxy-terminal regions which is missing in the previously published partial sequence. Sequencing of the intact and deglycosylated protein show that the glycosylation site is at Thr8. The amino acid composition accounts for a mass of 9623 Da, and the carbohydrate moeity was found to contribute 1490 Da. The biological activity of the porcine protein has been compared to recombinant human platelet factor 4 in an endothelial cell proliferation assay; both inhibit at a concentration giving half the maximal inhibition of 0.1 microM. Removal of the 19 amino-terminal residues carrying the carbohydrate moiety results in no change in the biological activity.

ATP citrate lyase is an enzyme involved in mammalian lipogenesis and cholesterogenesis. Inhibitors of the enzyme represent a potentially novel class of hypolipidemic agents. Citric acid analogues 5-16 bearing electrophilic and latent electrophilic substituents were synthesized and evaluated as irreversible inhibitors of the enzyme. The design of these agents was based on the classical enzymatic mechanism where an active-site nucleophile (thiol) was believed to be critically involved in catalysis. Reversible inhibition (Ki's ranging from ca. 20 to 500 microM) was observed for compounds 5, 10, and 12-16. Compounds 6-9 and 11 had no appreciable affinity for enzyme (Ki > 1 mM). Time-dependent inactivation of the enzyme by 5-16 was not detected following long incubation times (> 1 h, 37 degrees C) at 2 mM inhibitor concentrations.

Interleukin-8 is the most extensively characterised member of the structurally related chemotactic and pro-inflammatory proteins collectively called chemokines. It binds to two closely related members of the seven transmembrane chemokine receptor family found on a variety of leukocyte cell types. In order to study the interaction of interleukin-8 with its receptors, and their distribution, we have produced a fluorescently labelled protein as an alternative to the radioactive 125I-interleukin-8 ligand. Interleukin-8 is naturally produced as two forms, a 72-residue polypeptide by monocytes and a 77-residue form produced by endothelial cells which has an extension of five amino acids at the amino terminal. Both forms are active at nanomolar concentrations, implying that chemical modification to the amino terminus of the 72-residue form will not destroy activity. The 72-residue interleukin-8 sequence starts with a serine residue, which can be oxidised under mild conditions to give a reactive glyoxylyl function which is then reacted with a nucleophilic fluorescein derivative. The site-specifically labelled protein was easily isolated by reverse-phase HPLC. The dissociation constant of the fluorescently labelled interleukin-8 from its receptors on neutrophils was measured by displacement of 125I-interleukin-8 and found to be 10 nM compared to 1 nM for the unmodified protein. The modified protein is highly active in in vitro bioassays using human neutrophils, giving an EC50 of 7 nM in chemotaxis and an EC50 of 0.62 nM for shape change. The binding of the fluorescent protein to neutrophils can also be measured by fluorescent automatic cell sorter (FACS) analysis, and can be competed by unlabelled interleukin-8. The amino-terminal modification of interleukin-8 has produced a reagent which is useful for the quantification of interleukin-8 receptor expression, and will also be useful in monitoring the fate of the ligand after receptor binding.

Chemokines are chemoattractant proteins that are involved in basal trafficking, as well as recruitment and activation of cells in inflammatory processes. They are widely believed to act through a haptotactic mechanism, whereby immobilised gradients of chemokines are established on the endothelial cell surface and in the extracellular matrix [1]. Chemokines are known to bind to glycosaminoglycans (GAGs) in vitro and in vivo [2-4]. This GAG binding has been shown to promote oligomerisation [4], which has been hypothesised to increase local concentrations of chemokine in vivo [5]. Additionally, oligomerisation could enhance presentation to receptors [4, 6]. We recently demonstrated the essential role of the chemokine/GAG interaction with respect to biological activity in vivo [7], using a RANTES/CCL5 variant where the basic amino acids on the 40s loop were mutated to alanine, considerably reducing the capacity for heparin binding [8]. The aim ofthe present study was to investigate the potential of [44AANA47]-RANTES to block chemokine mediated inflammation in vivo.

Phosphomannose isomerase (PMI) is essential for the production of yeast cell walls. An inhibitor which inhibits the fungal enzyme without altering the activity of the mammalian enzyme would be a potential fungicidal agent, increasingly important in view of the increasing mortality from visceral mycoses in immunosuppressed patients. We have purified human, porcine, and Candida albicans enzymes 29,000-fold to homogeneity, and characterized their physical properties, as well as their kinetic parameters, inhibition constants, and pH dependences. Surprisingly, in view of the large differences between Pseudomonas aerugenosa and Saccharomyces cerevisiae PMI, the human and C. albicans enzymes are almost identical. We suggest therefore that species-selective inhibition of the fungal rather than mammalian enzyme may require molecules which bind away from the substrate binding pocket of the enzyme.

In our search for novel chemoattractant factors, we have purified a heparin-binding protein from porcine platelets which is a potent chemoattractant for human neutrophils. The protein has 80 amino acids and a molecular mass of 8597.5Da as measured by electrospray mass spectrometry. It has been characterised by amino acid sequencing and shown to have highest identity to members of the human platelet basic-protein-family. Its N-terminal sequence is intermediate in length between the human connective-tissue-activating polypeptide III (CTAP-III) and neutrophil-activating polypeptide-2 (NAP-2). The porcine NAP-2/CTAP-III shows the classic CXC cysteine spacing found towards the N-terminus in the chemokine alpha family and contains the ELR motif which has been shown to be essential for neutrophil chemotaxis. We have isolated mRNA from porcine platelets and constructed a cDNA library containing 1.0 x 10(6) independent clones. Using probes based on the protein sequence we have isolated a full length-clone for this gene, with an open reading frame containing 119 amino acids. Despite overall similarity between the human and porcine proteins, the N-terminal region is almost completely different between the two species, with only two identical amino acids. The proteolytic cleavage sites required for processing of human platelet basic protein are completely missing in the porcine homologue, implying a different processing pathway or mechanism. The porcine protein is capable of agonizing certain effects of both NAP-2 and CTAP-III when incubated with human cells indicating that the same porcine protein may be involved in both processes.

Interleukin-5 (IL-5) is a lineage-specific cytokine for eosinophilpoiesis and plays an important part in diseases associated with increased eosinophils, such as asthma. Human IL-5 is a disulphide-linked homodimer with 115 amino-acid residues in each chain. The crystal structure at 2.4 A resolution reveals a novel two-domain structure, with each domain showing a striking similarity to the cytokine fold found in granulocyte macrophage and macrophage colony-stimulating factors, IL-2 (ref. 5), IL-4 (ref. 6), and human and porcine growth hormones. IL-5 is unique in that each domain requires the participation of two chains. The IL-5 structure consists of two left-handed bundles of four helices laid end to end and two short beta-sheets on opposite sides of the molecule. Surprisingly, the C-terminal strand and helix of one chain complete a bundle of four helices and a beta-sheet with the N-terminal three helices and one strand of the other chain. The structure of IL-5 provides a molecular basis for the design of antagonists and agonists that would delineate receptor recognition determinants critical in signal transduction. This structure determination extends the family of the cytokine bundle of four helices and emphasizes its fundamental significance and versatility in recognizing its receptor.

Interleukin-5 (IL-5) plays a key role in the proliferation and differentiation of eosinophils. To aid the solution of the crystallographic three-dimensional structure, we have expressed large quantities of recombinant human IL-5 (hIL-5) in a methionine auxotroph strain of Escherichia coli (DL41) grown on an enriched seleno-DL-methionine-containing medium. Cell densities of A650 = 10 have been achieved. The selenomethionyl-labelled hIL-5 (Se-hIL-5) has been purified and found to contain 3.6 selenium atoms/dimer, and 0.4 methionine residues/dimer. In a B-cell growth factor assay, the Se-hIL-5 is significantly more active than the non-labelled hIL-5. Electrospray mass spectrometry shows two major peaks, with relative molecular masses of 26,326 +/- 6 and 26,280 +/- 8 corresponding to the 4Se and 3Se/1S forms of hIL-5. Unlike the methionine-containing hIL-5, the N-terminal selenomethionine is neither oxidised nor carbamoylated and can only be resolved into two species in isoelectric focusing gel electrophoresis. Se-hIL-5 crystallises in the same space group and unit cell as hIL-5. Difference Fourier calculations identify two of the selenomethionines corresponding to Met107 in the dimer. However, the N-terminal is disordered in the crystal, and the N-terminal selenomethionines are not resolved in the difference Fourier.

Phosphomannose isomerase (PMI) from Saccharomyces cerevisiae is a zinc-dependent metalloenzyme. Besides its role in catalysis, zinc is also a potent inhibitor of the enzyme. The inhibition is competitive with the substrate mannose 6-phosphate, with Kis = 6.4 microM in 50 mM Tris-HCl buffer, pH 8.0, at 37 degrees C. This inhibition constant is 4 orders of magnitude smaller than for group II divalent cations, indicating that the binding is not primarily electrostatic. Micromolar inhibition is also observed with ions of the other metals of the electronic configuration d10. Under identical conditions, cadmium is a predominantly competitive inhibitor with Kis = 19.5 microM. Inhibition by mercury is predominantly competitive with Kis = 6.0 microM but shows a hyperbolic Dixon plot. Theorell-Yonetani double-inhibition analysis shows that zinc and cadmium ions are mutually exclusive inhibitors against mannose 6-phosphate. However, analysis of zinc and mercury double inhibition shows that they can simultaneously bind in the mannose 6-phosphate binding pocket, with only a small mutual repulsion. Inhibition of the enzyme by cadmium and zinc ions is strongly pH dependent with pKa = 9.2 for cadmium and one pKa at 6.6 and two at 8.9 for zinc. The inhibitory species are the monohydroxide forms, Zn(OH)+ and Cd(OH)+. However, inhibition by mercury is relatively pH-independent, consistent with the neutral Hg(OH)2 being the inhibitory species. In all three cases, the metal ion binding causes a conformational change in the enzyme as judged by tryptophan fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Salt and water balance and vasopressin secretion were measured in three colonies of Sprague-Dawley rats. Although sodium and water retention were similar between the groups, there were marked differences in both the rate and diurnal pattern of intake and excretion. Animals housed under semi-barrier conditions showed a lower basal plasma vasopressin concentration but were more sensitive to physiological stimuli. However, since pathogenic status and environmental conditions cannot entirely explain these results, genetic variation is likely to be a contributory factor.

A powerful chemical modification procedure has been developed to define determinants of DNA recognition by the p50 subunit of NF-kappa B. Differential labelling with [14C] iodoacetate has identified a conserved cysteine residue, Cys62, that was protected from modification by the presence of an oligonucleotide containing the specific recognition site of the protein. To determine the importance of this cysteine residue, each of the conserved cysteines in p50 was changed to serine and the DNA binding properties of the mutant proteins determined. Scatchard analysis indicated that the C62S mutant bound to its DNA recognition site with a 10-fold larger dissociation constant than the wild type protein, while the other two mutants bound with an intermediate affinity. Dissociation rate constant measurements correlated well with the dissociation constants for the wild type, C119S, and C273S p50 proteins, whereas the p50 C62S-DNA complex dissociated anomalously quickly. Competition analyses with oligonucleotide variants of the DNA recognition site and nonspecific E. coli DNA revealed that the C62S p50 mutant had an altered DNA binding site specificity and was impaired in its ability to discriminate between specific and non-specific DNA. Thus the sulphydryl group of Cys62 is an important determinant of DNA recognition by the p50 subunit of NF-kappa B.