Host defense peptides such as defensins are components of innate immunity and have retained antibiotic activity throughout evolution. Their activity is thought to be due to amphipathic structures, which enable binding and disruption of microbial cytoplasmic membranes. Contrary to this, we show that plectasin, a fungal defensin, acts by directly binding the bacterial cell-wall precursor Lipid II. A wide range of genetic and biochemical approaches identify cell-wall biosynthesis as the pathway targeted by plectasin. In vitro assays for cell-wall synthesis identified Lipid II as the specific cellular target. Consistently, binding studies confirmed the formation of an equimolar stoichiometric complex between Lipid II and plectasin. Furthermore, key residues in plectasin involved in complex formation were identified using nuclear magnetic resonance spectroscopy and computational modeling.

The spatial organization of Pseudomonas aeruginosa and Staphylococcus aureus in chronic wounds was investigated in the present study. Wound biopsy specimens were obtained from patients diagnosed as having chronic venous leg ulcers, and bacterial aggregates in these wounds were detected and located by the use of peptide nucleic acid-based fluorescence in situ hybridization and confocal laser scanning microscopy (CLSM). We acquired CLSM images of multiple regions in multiple sections cut from five wounds containing P. aeruginosa and five wounds containing S. aureus and measured the distance of the bacterial aggregates to the wound surface. The distance of the P. aeruginosa aggregates to the wound surface was significantly greater than that of the S. aureus aggregates, suggesting that the distribution of the bacteria in the chronic wounds was nonrandom. The results are discussed in relation to our recent finding that swab culturing techniques may underestimate the presence of P. aeruginosa in chronic wounds and in relation to the hypothesis that P. aeruginosa bacteria located in the deeper regions of chronic wounds may play an important role in keeping the wounds arrested in a stage dominated by inflammatory processes.

Alanine scanning mutagenesis has been used to identify specific side chains of insulin which strongly influence binding to the insulin receptor. A total of 21 new insulin analog constructs were made, and in addition 7 high pressure liquid chromatography-purified analogs were tested, covering alanine substitutions in positions B1, B2, B3, B4, B8, B9, B10, B11, B12, B13, B16, B17, B18, B20, B21, B22, B26, A4, A8, A9, A12, A13, A14, A15, A16, A17, A19, and A21. Binding data on the analogs revealed that the alanine mutations that were most disruptive for binding were at positions TyrA19, GlyB8, LeuB11, and GluB13, resulting in decreases in affinity of 1,000-, 33-, 14-, and 8-fold, respectively, relative to wild-type insulin. In contrast, alanine substitutions at positions GlyB20, ArgB22, and SerA9 resulted in an increase in affinity for the insulin receptor. The most striking finding is that B20Ala insulin retains high affinity binding to the receptor. GlyB20 is conserved in insulins from different species, and in the structure of the B-chain it appears to be essential for the shift from the ␣-helix B8 -B19 to the ␤-turn B20 -B22. Thus, replacing GlyB20 with alanine most likely modifies the structure of the B-chain in this region, but this structural change appears to enhance binding to the insulin receptor.Insulin mediates its effects by binding to the insulin receptor in the plasma membrane of target cells. The molecular mechanisms for insulin interaction with the receptor are not fully understood. The crystal structure of the insulin molecule has been known for more than 25 years (1), but it remains an open question whether the structure of insulin that binds to the receptor is similar to the crystal structure. Until the structure of bound insulin and the side chains that are actually involved in binding is identified by co-crystallization of the receptor and ligand, more information about the binding domain on insulin can be obtained using mutational approaches.The binding domain of the insulin molecule has been studied by investigating receptor binding of a number of insulins from different animal species as well as chemically modified and more recently genetically engineered insulins (2-4). These studies have provided experimental support for a model in which invariant residues from both A and B chains form a surface that binds to the insulin receptor. The putative binding domain comprises a number of residues overlapping the dimerforming surface (ValB12, TyrB16, GlyB23, PheB24, PheB25, TyrB26, GlyA1, GlnA5, TyrA19, and AsnA21) and some of the residues buried beneath the COOH terminus of the B-chain (IleA2, ValA3, GluA4) (2). Cross-linking studies with an azidophenylalanine-substituted analog have shown that one of these residues, PheB25, comes into close proximity to the insulin receptor upon binding (5).Recently, a second binding site has been proposed, involving residues LeuA13 and LeuB17 (6, 7). A biphasic binding reaction involving this second binding site could explain the negative cooperativity phenomen...

We have synthesized insulins acylated by fatty acids in the epsilon-amino group of LysB29. Soluble preparations can be made in the usual concentration of 600 nmol/ml (100 IU/ml) at neutral pH. The time for 50% disappearance after subcutaneous injection of the corresponding TyrA14(125I)-labelled insulins in pigs correlated with the affinity for binding to albumin (r = 0.97), suggesting that the mechanism of prolonged disappearance is binding to albumin in subcutis. Most protracted was LysB29-tetradecanoyl des-(B30) insulin. The time for 50% disappearance was 14.3 +/- 2.2 h, significantly longer than that of Neutral Protamine Hagedorn (NPH) insulin, 10.5 +/- 4.3 h (p < 0.001), and with less inter-pig variation (p < 0.001). Intravenous bolus injections of LysB29-tetradecanoyl des-(B30) human insulin showed a protracted blood glucose lowering effect compared to that of human insulin. The relative affinity of LysB29-tetradecanoyl des-(B30) insulin to the insulin receptor is 46%. In a 24-h glucose clamp study in pigs the total glucose consumptions for LysB29-tetradecanoyl des-(B30) insulin and NPH were not significantly different (p = 0.88), whereas the times when 50% of the total glucose had been infused were significantly different, 7.9 +/- 1.0 h and 6.2 +/- 1.3 h, respectively (p < 0.04). The glucose disposal curve caused by LysB29-tetradecanoyl des-(B30) insulin was more steady than that caused by NPH, without the pronounced peak at 3 h. Unlike the crystalline insulins, the soluble LysB29-tetradecanoyl des-(B30) insulin does not elicit invasion of macrophages at the site of injection. Thus, LysB29-tetradecanoyl des-(B30) insulin might be suitable for providing basal insulin in the treatment of diabetes mellitus.

Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are both from the same subgroup of receptor tyrosine kinases that exist as covalently bound receptor dimers at the cell surface. For both IR and IGF-IR, the most described forms are homodimer receptors. However, hybrid receptors consisting of one-half IR and one-half IGF-IR are also present at the cell surface. Two splice variants of IR are expressed that enable formation of two isoforms of the IGF-IR/IR hybrid receptor. In this study, these two splice variants of hybrid receptors were studied with respect to binding affinities of insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II). Unlike previously published data, in which semipurified receptors have been studied, we found that the two hybrid receptor splice variants had similar binding characteristics with respect to insulin, IGF-I, and IGF-II binding. We studied both semipurified and purified hybrid receptors. In all cases we found that IGF-I had at least 50-fold higher affinity than insulin, irrespective of the splice variant. The binding characteristics of insulin and IGF-I to both splice variants of the hybrid receptors were similar to classical homodimer IGF-IR.

In a controlled, prospective, randomized investigation, started in 1974, 118 patients with supratentorial astrocytoma Grade III--IV were divided into three groups. Groups 1 and 2 received 45 Gy postoperatively to the whole supratentorial brain. Bleomycin in 15-mg doses and a total dose of 180 mg or placebo was given intravenously three times a week, one hour prior to radiotherapy, during weeks 1, 2, 4 and 5. Group 3 received conventional care but no radiotherapy or chemotherapy. Median survival rates of patients were 10.8 months in Groups 1 and 2, and 5.2 months in Groups 3, a statistically significant difference. With regard to performance, the patients in Group 3 deteriorated faster than patients in Groups 1 and 2. Bleomycin had no positive or negative influence on survival.

To identify the region(s) of the insulin receptor and the insulin-like growth factor I (IGF-I) receptor responsible for ligand specificity (high-affinity binding), expression vectors encoding soluble chimeric insulin/IGF-I receptors were prepared. The chimeric receptors were expressed in mammalian cells and partially purified. Binding studies revealed that a construct comprising an IGF-I receptor in which the 68 N-terminal amino acids of the insulin receptor a-subunit had replaced the equivalent IGF-I receptor segment displayed a markedly increased affinity for insulin. In contrast, the corresponding IGF-I receptor sequence is not critical for high-affinity IGF-I binding. It is shown that part of the cysteine-rich domain determines IGF-I specificity. We have previously shown that exchanging exons 1, 2, and 3 of the insulin receptor with the corresponding IGF-I receptor sequence results in loss of high affinity for insulin and gain ofhigh affinity for IGF-I. Consequently, it is suggested that the ligand specificities of the two receptors (i.e., the sequences that discriminate between insulin and IGF-I) reside in different regions of a binding site with common features present in both receptors.The polypeptide hormone insulin plays an essential role in metabolic regulation (1-3). It exerts its physiological effects through its interaction with a specific high-affinity receptor, which is an integral cell surface membrane glycoprotein with a relative molecular mass of about 400 kDa (4-6). The receptor is a disulfide-linked heterotetramer, made up of two a-and two B-subunits (7-12). Its ligand binding domains are formed by the extracellular a-subunits, whereas the /3-subunits consist of a short extracellular domain, a single transmembrane segment, and an intrinsic intracellular tyrosine protein kinase domain involved in signal transduction (13-15).The insulin-like growth factor I (IGF-I) receptor shows extensive similarity to the insulin receptor in amino acid sequence, domain structure, and signaling mechanism (11,16). Although insulin and IGF-I are very similar in amino acid sequence, they bind only weakly to the receptor for the other hormone (17,18).Ligand binding affinity and specificity are central to receptor activation, regulation, and function. Chimeric molecules comprising parts of related receptors have been useful for elucidating relationships between structure and specificity in several systems (e.g., refs. 19-21). In a previous study we showed that it is possible to convert the insulin receptor into a receptor with high affinity for IGF-I and low affinity for insulin by substituting an insulin receptor cDNA fragment (exons 1, 2, and 3) with that for the corresponding . This suggested to us that the ligand specificity of both receptors resides in the same region. Here, we extend this approach to a more detailed analysis of the ligand specificity of this receptor family. The first 68 N-terminal amino acids of the insulin receptor are found to determine insulin specificity. The corresponding 62 N-t...

Insulin is thought to elicit its effects by crosslinking the two extracellular ␣-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.I nsulin is one of the most studied peptide hormones because of its importance in maintaining glucose homeostasis. This 51-aa hormone is very well characterized with regard to its structure, both in crystal form and in solution. The insulin receptor (IR) is a transmembrane ␣ 2 ␤ 2 glycoprotein whose intracellular tyrosine kinase domain is activated by binding of insulin, leading to a cascade of intracellular signaling events. The kinase domain of the IR (1) and an extracellular fragment of the related receptor for insulin-like growth factor I (IGF-IR; ref. 2) have been crystallized, but the structure of the insulin binding domain of the IR is not known, and the mechanism for the transmission of a signal through its transmembrane domain is not well understood. A model for the binding and activation has been proposed in which insulin uses two different sites on its surface to crosslink the two ␣-subunits of the IR, thus inducing a conformational change that activates the receptor (refs. 3 and 4; Fig. 1).In a previous report (5), we panned random, highly diverse peptide display libraries against the IR. By using this approach, we identified a large number of peptides binding to the IR and competing for insulin binding with micromolar or submicromolar affinity, although these peptides had no sequence homology with insulin. These peptides bound to two discrete hotspots on the receptor (designated site 1 and site 2), and these hotspots appeared to correspond to the two contact sites involved in insulin binding predicted by the crosslinking model (ref. 3 and J.B., unpublished results). At least two different sequence motifs were found for site 1 peptides, and some of these were full agonists but of low affinity. Other site 1 peptides were antagonists, whereas site 2 peptides were either antagonists or inactive. The mechanism behind the agonism of the site 1 peptides is not known, but it has been speculated that site 1 binding may be important for receptor activation, whereas the role of the site 2 interaction may be more related to affinity and selectivity. In addition to these two families of peptides, a third group was identified, but no further work has been done on this group. In the present work, we have used site 1 and site 2 peptides as building blocks ...