Nonpeptidic, monocharged, cell permeable ligands for the p56lck SH2 domain.

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.

Highly potent and selective peptide-based inhibitors of the hepatitis C virus serine protease: towards smaller inhibitors.

Structure-activity studies on a hexapeptide N-terminal cleavage product of a dodecamer substrate led to the identification of very potent and highly specific inhibitors of the HCV NS3 protease/NS4A cofactor peptide complex. The largest increase in potency was accomplished by the introduction of a (4R)-naphthalen-1-yl-4-methoxy substituent to the P2 proline. N-Terminal truncation resulted in tetrapeptides containing a C-terminal carboxylic acid, which exhibited low micromolar activity against the HCV serine protease.

Antibiotic MIC/MBC analysis of Bacillus-based commercial insecticides: use of bioreduction and DNA-based assays.

Minimum inhibitory concentration (MIC) assays, monitored by colony counts, growth (turbidity) and bioreduction of non-toxic XTT [2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide, inner salt], were used to assess the performance of several types of antibiotics against: (1) commercial BT products made from scale-up sporulation phase cultures of Bacillus thuringiensis subsp israelensis (Bti) and subsp kurstaki (Btk); (2) vegetative cells derived from these BT products; and (3) Gram-positive and Gram-negative bacteria used as controls. The XTT-kinetic assay improved sensitivity and early reading of MIC breakpoints. The conventional colony count method for determining minimal bactericidal concentration (MBC) was used to validate a multi-sample dot-blot assay in which organisms in individual MIC assays are trapped free of residual antibiotic and their viability is estimated by in situ conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] to insoluble formazan. Tolerance (MBC/MIC) for most antibiotics was low (

Ligands for the tyrosine kinase p56lck SH2 domain: discovery of potent dipeptide derivatives with monocharged, nonhydrolyzable phosphate replacements.

p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.

A randomized controlled trial of nonpharmacologic approaches for relief of low back pain during labor.

BACKGROUND: Low back pain is common during labor. Our randomized controlled trial compared the effectiveness of 3 nonpharmacologic approaches for relief of back pain. METHODS: A total of 34 women suffering from low back pain during labor were randomly assigned to receive 1 of 3 treatments: (1) intracutaneous sterile water injections (ISW); (2) transcutaneous electrical nerve stimulation (TENS); and (3) standard care, including back massage, whirlpool bath, and liberal mobilization. Women self-evaluated both intensity and affective dimensions of pain using visual analog scales. Their evaluations of control and satisfaction were assessed using adapted versions of the Labour Agentry Scale and the Labor and Delivery Satisfaction Index. RESULTS: Women in the ISW group rated the intensity and unpleasantness of pain during the experimental period significantly lower than women in the standard care group or the TENS group, (P = .001 and P = .003, respectively). Similar results were observed for intensity (P = .01) and unpleasantness (P = .03) of pain assessed just before delivery or request for an epidural. Mean pain intensity at 15 and 60 minutes after randomization was significantly reduced in the ISW group compared with the 2 other groups. There was no significant difference in the 3 groups in the level of control and satisfaction with labor and delivery, but less women in the ISW group indicated that they would like to receive the same treatment for back pain during another delivery. CONCLUSIONS: Intracutaneous sterile water injections are more effective than standard care (back massage, bath, and mobilization) or transcutaneous electrical nerve stimulation for relieving low back pain during labor.

Phosphotyrosine-containing dipeptides as high-affinity ligands for the p56lck SH2 domain.

Src homology-2 (SH2) domains are noncatalytic motifs containing approximately 100 amino acid residues that are involved in intracellular signal transduction. The phosphotyrosine-containing tetrapeptide Ac-pYEEI binds to the SH2 domain of p56lck (Lck) with an affinity of 0.1 microM. Starting from Ac-pYEEI, we have designed potent antagonists of the Lck SH2 domain which are reduced in peptidic character and in which the three carboxyl groups have been eliminated. The two C-terminal amino acids (EI) have been replaced by benzylamine derivatives and the pY + 1 glutamic acid has been substituted with leucine. The best C-terminal fragment identified, (S)-1-(4-isopropylphenyl)ethylamine, binds to the Lck SH2 domain better than the C-terminal dipeptide EI. Molecular modeling suggests that the substituents at the 4-position of the phenyl ring occupy the pY + 3 lipophilic pocket in the SH2 domain originally occupied by the isoleucine side chain. This new series of phosphotyrosine-containing dipeptides binds to the Lck SH2 domain with potencies comparable to that of tetrapeptide 1.

Studies on the C-terminal of hexapeptide inhibitors of the hepatitis C virus serine protease.

Replacement of the C-terminal carboxylic acid functionality of peptide inhibitors of hepatitis C virus (HCV) NS3 protease (complexed with NS4A peptide cofactor) by activated carbonyl groups does not produce any substantial increase in potency. These latter inhibitors also inhibit a variety of other serine and cysteine proteases whereas the carboxylic acids are specific. Norvaline was identified as a chemically stable replacement for the P1 residue of Ac-DDIVPC-OH which was also compatible with activated carbonyl functionalities.

Ellipsometric study of thermally evaporated germanium thin film.

The optical properties of thermally evaporated germanium thin films in the spectral range 0.3-1.7 mum were studied with spectroscopic ellipsometry. The microstructure of these films, including their crystallinity, density, surface morphology, and surface oxidation, was analyzed with x-ray diffraction, Rutherford backscattering spectrometry, atomic force microscopy (AFM), and Auger electron spectrometry (AES). Parameters such as the surface roughness and surface-oxidation-layer thickness, derived from AFM and AES measurements, were incorporated into our optical model. The complex index of refraction (n and k) of the films was determined throughout the above spectral range and compared with that of single-crystal germanium.

Refractive-index measurements of moderately reflecting substrates using a wedged film technique.

A method is described whereby the complex index of refraction can be determined for a strongly absorbing substrate material by measurement of reflectance maxima and minima at a given wavelength, as a function of the thickness of a wedged nonabsorbing thin film layer deposited on the substrate. The procedure for determination of the complex index n - ik employs the reflectance extremum afd correlated film thickness to construct graphically the complex reflectance amplitude of the substrate material. Subsequently, the Fresnel equations for reflectance amplitude can be used to calculate the values of n and k. Examples of the successful use of the method to determine the far-IR indices of fused silica and sapphire are given. The limitations of the method are discussed, and examples of the expected errors for n and k for the two specific cases calculated are given.

Effects of dilution on a six-element synthetic aperture.

An investigation was made of the effects of dilution on a six-element synthetic aperture. The characteristics of both the spread function and the transfer function were obtained for several degrees of dilution. It is shown that small degrees of dilution result in a significant gain over the performance of a single element, whereas more dilute apertures are inferior for direct imagery.