Bacterial membrane disrupting dodecapeptide SC4 improves survival of mice challenged with Pseudomonas aeruginosa.

Background: Dodecapeptide SC4 is a broad-spectrum bactericidal agent that functions by disintegrating bacterial membranes and neutralizing endotoxins. For insight into which SC4 amino acids are functionally important, we assessed Gram-negative bactericidal effects in structure-activity relationship experiments. Subsequently, SC4 was tested in a murine bacteremia model to combine and compare the efficacy with Zosyn, a first-line antibiotic against Pseudomonas aeruginosa (P.

Long-term functional improvement and gene expression changes after bone marrow-derived multipotent progenitor cell transplantation in myocardial infarction.

The study examined the long-term outcome of cardiac stem cell transplantation in hearts with postinfarction left ventricular (LV) remodeling. Myocardial infarction (MI) was created by ligating the first and second diagonal branches of the left anterior descending coronary artery in miniature swine. Intramyocardial injections of 50 million LacZ-labeled bone marrow-derived multipotent progenitor cells (MPC) were performed in the periscar region (Cell, n = 7) immediately after MI, whereas, in control animals (Cont, n = 7), saline was injected.

Metastin (KiSS-1) mimetics identified from peptide structure-activity relationship-derived pharmacophores and directed small molecule database screening.

Metastin, also known as KiSS-1, the cognate ligand for the metastin receptor GPR54, is a peptide known to dramatically reduce metastasis in experimental models. Despite this, there is no reported structure for metastin nor any small molecule modulators of metastin function that could be used either clinically or experimentally. Here we report the NMR solution structure of a 13-residue metastin peptide in a membrane-like environment (SDS micelles) and find it to have a relatively stable helix conformation from residues 7 to 13.

Profound bioenergetic abnormalities in peri-infarct myocardial regions.

Regions of myocardial infarct (MI) are surrounded by a border zone (BZ) of normally perfused but dysfunctional myocardium. Although systolic dysfunction has been attributed to elevated wall stress in this region, there is evidence that intrinsic abnormalities of contractile performance exist in BZ myocardium. This study examined whether decreases of high-energy phosphates (HEP) and mitochondrial F(1)F(0)-ATPase (mtATPase) subunits typical of failing myocardium exist in BZ myocardium of compensated postinfarct remodeled hearts.

A simple method to measure 13CH2 heteronuclear dipolar cross-correlation spectral densities.

Here, we report a method to simultaneously determine CH2 cross-correlation spectral densities and T1 relaxation times in the laboratory and rotating frames. To accomplish this, we have employed an indirect approach that is based on measurement of differences in relaxation rates acquired with and without cross-correlation terms. The new method, which can be employed using multidimensional NMR and standard relaxation pulse sequences, is validated experimentally by investigation of a selectively 13C-enriched hexadecapeptide and the uniformly 13C-enriched immunoglobulin-binding domain of streptococcal protein G (GB1).

Molecular biology of myocardial recovery.

The use of LVADs that leads to a dramatic mechanical and hemodynamic unloading of the failing left ventricle offers a unique opportunity to investigate the mechanisms of remodeling and reverse remodeling. Although it is being increasingly realized that the LVAD unloading results in regression of hypertrophy and improvement of myocyte function and LV geometry, the cellular and molecular mechanisms responsible for these beneficial effects remain undefined. The favorable alterations in geometry that occur in parallel fashion at the organ, cellular, and molecular levels are most likely caused by the reduced LV wall stress/stretch as a consequence of the mechanical support provided by LVAD.

Myocardial oxygenation and high-energy phosphate levels during KATP channel blockade.

Inhibition of ATP-sensitive K+ (KATP) channel activity has previously been demonstrated to result in coronary vasoconstriction with decreased myocardial blood flow and loss of phosphocreatine (PCr). This study was performed to determine whether the high-energy phosphate abnormality during KATP channel blockade can be ascribed to oxygen insufficiency. Myocardial blood flow and oxygen extraction were measured in open-chest dogs during KATP channel blockade with intracoronary glibenclamide, whereas high-energy phosphates were examined with 31P magnetic resonance spectroscopy (MRS), and myocardial deoxymyoglobin (Mb-delta) was determined with 1H MRS.

Beta-sheet is the bioactive conformation of the anti-angiogenic anginex peptide.

Anginex is a designed peptide 33mer that functions as a cytokine-like agent to inhibit angiogenesis. Although this short linear peptide has been shown by NMR and CD to form a nascent beta-sheet conformation in solution, the actual bioactive structure formed upon binding to its receptor on the surface of endothelial cells could be quite different. By using a series of double-cysteine disulphide-bridged analogues, we provide evidence in the present study that the beta-sheet is in fact the bioactive conformation of anginex.

Protein dynamics using frequency-dependent order parameters from analysis of NMR relaxation data.

A novel approach is described to analyze NMR relaxation data on proteins. This method introduces the frequency-dependent order parameter, S(2)(omega), in order to estimate contributions to the generalized order parameter S(2) from different motional frequencies occurring on the picosecond to nanosecond time scales. S(2)(omega) is defined as the sum of a specified set of weighting coefficients from the Lorentzian expansion of the spectral density function.

Mechanisms of cytokine induced NO-mediated cardiac fibroblast apoptosis.

This study examined the role of nitric oxide (NO) in cytokine-induced apoptosis in adult cardiac fibroblasts (CFbs). In cultured adult rat CFbs, IL-1beta (5 ng/ml), but not interferon-gamma (10 ng/ml) or tumor necrosis factor-alpha (10 ng/ml), induced inducible NO synthase (iNOS) expression and NO production that was associated with an increase in caspase-3 activity and apoptotic cell death. Apoptotic frequency was reduced by the iNOS inhibitor S-methylisothiourea (3 x 10(-5) M).

A new approach to visualizing spectral density functions and deriving motional correlation time distributions: applications to an alpha-helix-forming peptide and to a well-folded protein.

A new approach to visualizing spectral densities and analyzing NMR relaxation data has been developed. By plotting the spectral density function, J(omega), as F(omega)=2 omega J(omega) on the log-log scale, the distribution of motional correlation times can be easily visualized. F(omega) is calculated from experimental data using a multi-Lorentzian expansion that is insensitive to the number of Lorentzians used and allows contributions from overall tumbling and internal motions to be separated without explicitly determining values for correlation times and their weighting coefficients.

Conformational exchange on the microsecond time scale in alpha-helix and beta-hairpin peptides measured by 13C NMR transverse relaxation.

13C-NMR relaxation experiments (T(1), T(2), T(1)(rho), and NOE) were performed on selectively enriched residues in two peptides, one hydrophobic staple alpha-helix-forming peptide GFSKAELAKARAAKRGGY and one beta-hairpin-forming peptide RGITVNGKTYGR, in water and in water/trifluoroethanol (TFE). Exchange contributions, R(ex), to spin-spin relaxation rates for (13)C(alpha) and (13)C(beta) groups were derived and were ascribed to be mainly due to peptide folding-unfolding. To evaluate the exchange time, tau(ex), from R(ex), the chemical shift difference between folded and unfolded states, Deltadelta, and the populations of these states, p(i), were determined from the temperature dependence of (13)C chemical shifts.

Internal motional amplitudes and correlated bond rotations in an alpha-helical peptide derived from 13C and 15N NMR relaxation.

Peptide GFSKAELAKARAAKRGGY folds in an alpha-helical conformation that is stabilized by formation of a hydrophobic staple motif and an N-terminal capping box (Munoz V. Blanco FJ, Serrano L, 1995, Struct Biol 2:380-385). To investigate backbone and side-chain internal motions within the helix and hydrophobic staple, residues F2, A5, L7, A8, and A10 were selectively 13C- and 15N-enriched and NMR relaxation experiments were performed in water and in water/trifluoroethanol (TFE) solution at four Larmor frequencies (62.

Peptide internal motions on nanosecond time scale derived from direct fitting of (13)C and (15)N NMR spectral density functions.

NMR relaxation-derived spectral densities provide information on molecular and internal motions occurring on the picosecond to nanosecond time scales. Using (13)C and (15)N NMR relaxation parameters [T(1), T(2), and NOE] acquired at four Larmor frequencies (for (13)C: 62.5, 125, 150, and 200 MHz), spectral densities J(0), J(omega(C)), J(omega(H)), J(omega(H) + omega(C)), J(omega(H) - omega(C)), J(omega(N)), J(omega(H) + omega(N)), and J(omega(H) - omega(N)) were derived as a function of frequency for (15)NH, (13)C(alpha)H, and (13)C(beta)H(3) groups of an alanine residue in an alpha-helix-forming peptide.

Structure-function relationships in novel peptide dodecamerswith broad-spectrum bactericidal and endotoxin-neutralizing activities.

A series of designed peptide 33-mers (betapep peptides) areknown to be bactericidal [Mayo, Haseman, Ilyina and Gray (1998)Biochim. Biophys. Acta 1425, 81-92].

Heparin dodecasaccharide binding to platelet factor-4 and growth-related protein-alpha. Induction of a partially folded state and implications for heparin-induced thrombocytopenia.

alpha-Chemokines are known heparin-binding proteins. Here, a heparin dodecasaccharide (H12) was purified and used in NMR studies to investigate binding to growth-related protein-alpha (Gro-alpha) and to platelet factor-4-M2 (PF4-M2), an N-terminal chimera of PF4. Pulsed field gradient NMR was used to derive diffusion coefficients as the protein (monomer):H12 ratio was varied.

Designed beta-sheet-forming peptide 33mers with potent human bactericidal/permeability increasing protein-like bactericidal and endotoxin neutralizing activities.

Novel peptide 33mers have been designed by incorporating beta-conformation stabilizing residues from the beta-sheet domains of alpha-chemokines and functionally important residues from the beta-sheet domain of human neutrophil bactericidal protein (B/PI). B/PI is known for its ability to kill bacteria and to neutralize the action of bacterial endotoxin (lipopolysaccharide, LPS) which can induce septic shock leading to eventual death. Here, the goal was to make short linear peptides which demonstrate good beta-sheet folding and maintain bioactivity as in native B/PI.

NMR solution conformation of heparin-derived hexasaccharide.

The solution conformation of homogeneous, heparin-derived hexasaccharide (residues A, B, C, D, E, F) has been investigated by using 1H-NMR spectroscopy. Intra-ring conformations have been defined by J-coupling constants and inter-proton nuclear Overhauser effects (NOEs), and the orientation of one ring with respect to the other has been defined by inter-ring NOEs. NOE-based conformational modelling has been done by using the iterative relaxation matrix approach (IRMA), restrained energy minimization to refine structures and to distinguish between minor structural differences and equilibria between various intra-ring forms.

Alcohol consumption in the guinea pig is associated with reduced megakaryocyte deformability and platelet size.

Mild thrombocytopenia is common in alcoholic individuals. Ethanol appears to impair platelet production primarily by affecting the maturing megakaryocyte compartment. We recently showed that guinea pigs have an unusually large number of elongated platelet forms even under conditions of steady-state thrombopoiesis, and a portion of their mature (stage IV) megakaryocytes yield extremely long extensions on micropipette aspiration.

Resolution of pulmonary metastases with chemotherapy in a patient with placental site trophoblastic tumor.

Placental site trophoblastic tumor (PSTT), a rare variant of gestational trophoblastic disease, was first described in 1976. PSTT is usually seen in young women, generally treated by hysterectomy, and is associated with a 20% fatality rate. The development of metastases secondary to PSTT is associated with an extremely poor prognosis.

Serum levels of macrophage colony-stimulating factor in patients with ovarian cancer undergoing second-look laparotomy.

Objective: The purpose of this study was to evaluate the prognostic significance of macrophage colony-stimulating factor serum levels in patients with ovarian cancer undergoing second-look laparotomy.

Study Design: The presurgical serum levels of macrophage colony-stimulating factor from 33 consecutive patients with ovarian cancer undergoing second-look laparotomy were determined and compared with those of 50 healthy control subjects. Mean differences in groups were evaluated with the Student t test.