Conformational and ion binding properties of a cyclic octapeptide, cyclo (Ala-Leu-Pro-Gly)2.

The conformation and ion-binding characteristics of a cyclic octapeptide, cyclo (Ala-Leu-Pro-Gly)2, in a liphophilic solvent, acetonitrile, have been studied using CD and nmr spectroscopy. The peptide binds preferentially to divalent cations such as calcium, magnesium, and barium. The conformations of the free cyclic peptide and its calcium complex are very similar with well-defined beta- and gamma-turns. The cyclic peptide readily forms equimolar and possibly 2:1 (peptide:cation) complexes with divalent cations.     

Design of cyclic and d‐amino acids containing peptidomimetics for inhibition of protein‐protein interactions of HER2‐HER3

HER2 receptors are surface proteins belonging to the epidermal growth factor family of receptors. Their numbers are elevated in breast, lung, and ovarian cancers. HER2‐positive cancers are aggressive, have higher mortality rate, and have a poor prognosis. We have designed peptidomimetics that bind to HER2 and block the HER2‐mediated dimerization of epidermal growth factor family of receptors. Among these, a symmetrical cyclic peptidomimetic (compound 18 ) exhibited antiproliferative activity in HER2‐overexpressing lung cancer cell lines with IC₅₀ values in the nanomolar concentration range. To improve the stability of the peptidomimetic, d ‐amino acids were introduced into the peptidomimetic, and several analogs of compound 18 were designed. Among the analogs of compound 18 , compound 32 , a cyclic, d ‐amino acid‐containing peptidomimetic, was found to have an IC₅₀ value in the nanomolar range in HER2‐overexpressing cancer cell lines. The antiproliferative activity of compound 32 was also measured by using a 3D cell culture model that mimics the in vivo conditions. The binding of compound 32 to the HER2 protein was studied by surface plasmon resonance. In vitro stability studies indicated that compound 32 was stable in serum for 48 hours and intact peptide was detectable in vivo for 12 hours. Results from our studies indicated that 1 of the d ‐amino acid analogs of 18 , compound 32 , binds to the HER2 extracellular domain, inhibiting the phosphorylation of kinase of HER2.     

Development of co-dominant KASP markers co-segregating with Ug99 effective stem rust resistance gene <Emphasis Type="Italic">Sr26</Emphasis> in wheat

Stem rust of wheat, caused by Puccinia graminis f. sp. tritici (Pgt), is a threat to global food security due to its ability to cause total crop failures. The Pgt race TTKSK (Ug99) and its derivatives detected in East Africa carry virulence for many resistance genes present in modern cultivars. However, stem rust resistance gene Sr26 remains effective to all races of Pgt worldwide. Sr26 is carried on the Agropyron elongatum (syn. Thinopyrum ponticum) segment 6Ae#1L translocated to chromosome 6AL of wheat. In this study, a recombinant inbred line (RIL) population derived from a cross between the landrace Aus27969 and Avocet S, which carries Sr26, was used to develop co-dominant kompetitive allele-specific polymerase chain reaction (KASP) markers that co-segregate with Sr26. Four KASP markers (sunKASP_216, sunKASP_218, sunKASP_224 and sunKASP_225) were also shown to co-segregate with Sr26 in four additional RIL populations. When tested on Australian cultivars and breeding lines, these markers amplified alleles alternate to that linked with Sr26 in all cultivars known to lack this gene and Sr26-linked alleles in cultivars and genotypes known to carry Sr26. Genotypes WA-1 and WA-1/3*Yitpi carrying the shortest Sr26 translocation segment were positive only for markers sunKASP_224 and sunKASP_225. Our results suggest the four KASP markers are located on the original translocation and sunKASP_224 and sunKASP_225 are located on the shortened version. Therefore, sunKASP_224 and sunKASP_225 can be used for marker-assisted pyramiding of Sr26 with other stem rust resistance genes to achieve durable resistance in wheat.     

Regulation of myostatin expression is associated with growth and muscle development in commercial broiler and DMC muscle

Myostatin is a negative regulator of skeletal muscle growth. Muscle tissue is the largest tissue in the body and influences body growth. Commercial Avian broiler chickens are selected for high growth rate and muscularity. Daweishan mini chickens are a slow growing small-sized chicken breed. We investigated the relations between muscle (breast and leg) myostatin mRNA expression and body and muscle growth. Twenty chickens per breed were slaughtered at 0, 30, 60, 90, 120, and 150 days of age. Body and muscle weights were higher at all times in Avian chickens. Breast muscle myostatin expression was higher in Avian chickens than in Daweishan mini chickens at day 30. Myostatin expression peaked at day 60 in Daweishan mini chickens and expression remained higher in breast muscle. Daweishan mini chickens myostatin expression correlated positively with carcass weight, breast and leg muscle weight from day 0 to 60, and correlated negatively with body weight from day 90 to 150, while myostatin expression in Avian chickens was negatively correlated with carcass and muscle weight from day 90 to 150. The results suggest that myostatin expression is related to regulation of body growth and muscle development, with two different regulatory mechanisms that switch between days 30 and 60.     

Protein folding determinants: structural features determining alternative disulfide pairing in alpha- and chi/lambda-conotoxins

Alpha-conotoxins isolated from Conus venoms contain 11-19 residues and preferentially fold into the globular conformation that possesses a specific disulfide pairing pattern (C1-3, C2-4). We and others isolated a new family of chi-conotoxins (also called lambda conotoxins) with the conserved cysteine framework of alpha-conotoxins but with alternative disulfide pairing (C1-4, C2-3) resulting in the ribbon conformation. In both families, disulfide pairing and hence folding are important for their biological potency. By comparing the structural differences, we identified potential structural determinants responsible for the folding tendencies of these conotoxins. We examined the role of conserved proline in the first intercysteine loop and the conserved C-terminal amide on folding patterns of synthetic analogues of ImI conotoxin by comparing the isoforms with the regiospecifically synthesized conformers. Deamidation at the C-terminus and substitution of proline in the first intercysteine loop switch the folding pattern from the globular form of alpha-conotoxins to the ribbon form of chi/lambda-conotoxins. The findings are corroborated by reciprocal folding of CMrVIA chi/lambda-conotoxins. Substitution of Lys-6 from the first intercysteine loop of CMrVIA conotoxin with proline, as well as the inclusion of an amidated C-terminal shifted the folding preference of CMrVIA conotoxin from its native ribbon conformation toward the globular conformation. Binding assays of ImI conotoxin analogues with Aplysia and Bulinus acetylcholine binding protein indicate that both these substitutions and their consequent conformational change substantially impact the binding affinity of ImI conotoxin. These results strongly indicate that the first intercysteine loop proline and C-terminal amidation act as conformational switches in alpha- and chi/lambda-conotoxins.     

Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH.

The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide (1), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide-receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1-Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible beta-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first beta-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second beta-turn. Furthermore, the presence of a beta-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH-NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I beta-turn structure is also supported by CD spectral analysis. The cIBR peptide (1) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.     

Long-term survival in patients treated with ruxolitinib for myelofibrosis: COMFORT-I and -II pooled analyses

Background

Myelofibrosis (MF) is associated with a variety of burdensome symptoms and reduced survival compared with age-/sex-matched controls. This analysis evaluated the long-term survival benefit with ruxolitinib, a Janus kinase (JAK)1/JAK2 inhibitor, in patients with intermediate-2 (int-2) or high-risk MF.

Methods

This was an exploratory analysis of 5-year data pooled from the phase 3 COMFORT-I and -II trials. In both trials, patients could cross over to ruxolitinib from the control group (COMFORT-I, placebo; COMFORT-II, best available therapy). All continuing patients in the control groups crossed over to ruxolitinib by the 3-year follow-up. Overall survival (OS; a secondary endpoint in both trials) was evaluated using pooled intent-to-treat data from patients randomized to ruxolitinib or the control groups. OS was also evaluated in subgroups stratified by baseline anemia and transfusion status at week 24.

Results

A total of 528 patients were included in this analysis; 301 were originally randomized to ruxolitinib (COMFORT-I, n?=?155; COMFORT-II, n?=?146) and 227 to control (n?=?154 and n?=?73, respectively). The risk of death was reduced by 30% among patients randomized to ruxolitinib compared with patients in the control group (median OS, 5.3 vs 3.8 years, respectively; hazard ratio [HR], 0.70 [95% CI, 0.54–0.91]; P?=?0.0065). After correcting for crossover using a rank-preserving structural failure time (RPSFT) method, the OS advantage was more pronounced for patients who were originally randomized to ruxolitinib compared with patients who crossed over from control to ruxolitinib (median OS, 5.3 vs 2.3 years; HR [ruxolitinib vs RPSFT], 0.35 [95% CI, 0.23–0.59]). An analysis of OS censoring patients at the time of crossover also demonstrated that ruxolitinib prolonged OS compared with control (median OS, 5.3 vs 2.4 years; HR [ruxolitinib vs censored at crossover], 0.53 [95% CI, 0.36–0.78]; P?=?0.0013). The survival benefit with ruxolitinib was observed irrespective of baseline anemia status or transfusion requirements at week 24.

Conclusions

These findings support ruxolitinib treatment for patients with int-2 or high-risk MF, regardless of anemia or transfusion status. Further analyses will be important for exploring ruxolitinib earlier in the disease course to assess the effect on the natural history of MF.

Trial registration

ClinicalTrials.gov identifiers, NCT00952289 and NCT00934544.