NMR search for polymorphic phase transformations in chlorpropamide form-A at high pressures.

The high-pressure effects on chlorpropamide (C10H13ClN2O3S) form-A have been studied by 1H NMR spectroscopy at high pressures up to 800 MPa in the temperature range 90-300 K. A study of the NMR second moment and spin-lattice relaxation time has been completed by a calculation of the steric hindrances for molecular reorientations and simulations of the second moment of the NMR line by the Monte-Carlo method, which enabled a precise description of molecular dynamics in the compound studied. Reorientations of the methyl group, oscillations and reorientations of the chlorophenyl ring and reorientations of the propyl group have been revealed and respective activation parameters extracted. No phase transformation of the compound form-A has been detected.

Solution structure of the antiapoptotic protein bcl-2.

The structures of two isoforms of Bcl-2 that differ by two amino acids have been determined by NMR spectroscopy. Because wild-type Bcl-2 behaved poorly in solution, the structures were determined by using Bcl-2/Bcl-x(L) chimeras in which part of the putative unstructured loop of Bcl-2 was replaced with a shortened loop from Bcl-x(L). These chimeric proteins have a low pI compared with the wild-type protein and are soluble. The structures of the two Bcl-2 isoforms consist of 6 alpha-helices with a hydrophobic groove on the surface similar to that observed for the homologous protein, Bcl-x(L). Comparison of the Bcl-2 structures to that of Bcl-x(L) shows that although the overall fold is the same, there are differences in the structural topology and electrostatic potential of the binding groove. Although the structures of the two isoforms of Bcl-2 are virtually identical, differences were observed in the ability of the proteins to bind to a 25-residue peptide from the proapoptotic Bad protein and a 16-residue peptide from the proapoptotic Bak protein. These results suggest that there are subtle differences in the hydrophobic binding groove in Bcl-2 that may translate into differences in antiapoptotic activity for the two isoforms.

An approach for high-throughput structure determination of proteins by NMR spectroscopy.

An approach is described for rapidly determining protein structures by NMR that utilizes proteins containing 13C-methyl labeled Val, Leu, and Ile (delta1) and protonated Phe and Tyr in a deuterated background. Using this strategy, the key NOEs that define the hydrophobic core and overall fold of the protein are easily obtained. NMR data are acquired using cryogenic probe technology which markedly reduces the spectrometer time needed for data acquisition. The approach is demonstrated by determining the overall fold of the antiapoptotic protein, Bcl-xL, from data collected in only 4 days. Refinement of the Bcl-xL structure to a backbone rmsd of 0.95 A was accomplished with data collected in an additional 3 days. A distance analysis of 180 different proteins and structure calculations using simulated data suggests that our method will allow the global folds of a wide variety of proteins to be determined.

Quadrupolar and chemical shift tensors characterized by 2D multiple-quantum NMR spectroscopy

The present work discusses a new 2D NMR method for characterizing the principal values and relative orientations of the electric field gradient and the chemical shift tensors of half-integer quadrupolar sites. The technique exploits the different contributions that quadrupolar and shielding interactions impart on the evolution of multiple-quantum and of single-quantum coherences, in order to obtain 2D powder lineshapes that are highly sensitive to these nuclear spin coupling parameters. Different spinning variants of this experiment were assayed, but it was concluded that a static version can yield the highest sensitivity to the values of the principal components and to the relative geometries of the local coupling tensors. It was found that correlating the central transition evolution with the highest available order of the spin coherence was also helpful for maximizing this spectral information. Good agreement between data obtained on 87Rb (S = 32) and 59Co (S = 72) samples and ideal theoretical lineshape predictions of this experiment was obtained, provided that heterogeneities in the multiple-quantum excitation and conversion processes were suitably accounted by procedures similar to those described in the spin-(1/2) multiple-quantum NMR literature. Copyright 1999 Academic Press.

Solid and liquid phase 59Co NMR studies of cobalamins and their derivatives.

We describe the application of 59Co NMR to the study of naturally occurring cobalamins. Targets of these investigations included vitamin B12, the B12 coenzyme, methylcobalamin, and dicyanocobyrinic acid heptamethylester. These measurements were carried out on solutions and powders of different origins, and repeated at a variety of magnetic field strengths. Particularly informative were the solid-state central transition NMR spectra, which when combined with numerical line shape analyses provided a clear description of the cobalt coupling parameters. These parameters showed a high sensitivity to the type of ligands attached to the metal and to the crystallization history of the sample. 59Co NMR determinations also were carried out on synthetic cobaloximes possessing alkyl, cyanide, aquo, and nitrogenated axial groups, substituents that paralleled the coordination of the natural compounds. These analogs displayed coupling anisotropies comparable to those of the cobalamins, as well as systematic up-field shifts that can be rationalized in terms of their stronger binding affinity to the cobalt atom. Cobaloximes also displayed a higher regularity in the relative orientations of their quadrupole and shielding coupling tensors, reflecting a higher symmetry in their in-plane coordination. For the cobalamines, poor correlations were observed between the values measured for the quadrupole couplings in the solid and the line widths observed in the corresponding solution 59Co NMR resonances.

Preliminary experimental experiences with xenotransplantations.

In our xenotransplantation experiments has been a major aim to induce specific tolerance. The results from the meeting of the donor's and the recipient's immunocompetent systems already in the period of embryonic development and/or the influence of methylprednisolone are described.

Effect of some drugs, experimental stress and estrus on unstable and fixed conditioned alimentary motor reflexes in cats. Reproduction and stress. Part V.

Reproduction of fixed conditioned alimentary motor reflexes in long-time laboratory kept cats in the course of the year is remarkably influenced by the sexual cycle. Estrus, anestrus, gestation and deliveries act upon the reflex reproduction in a similar way like deliberate application of experimental stress of various intensity. The study of the antistress efficacy of drugs modifying psychological functions and mental states in the method of reproduction of fixed alimentary motor reflexes under stress must specify the actual effect of experimental stress. Experimental sensory stress represented by intermittent sound of 98 dB and intermittent light (0.6 joules) acting with simultaneous frequency of 6 Hz was found to generally impair reflex reproduction over the whole period of twelve months. Correct response latencies were significantly prolonged and incorrect motor reactions significantly increased. If reflex reproduction was impaired due to the sexual cycle, simultaneous sensory stress still aggravates the impairment. The definition of the actual efficacy of experimental stressing thus necessarily requires regular control experiments in the course of the year with reproduction influenced neither by stress nor drugs at least at forthightly intervals.

Effect of some drugs, experimental stress and estrus on unstable and fixed conditioned alimentary motor reflexes in cats. Meclophenoxate, chlorprothixen, caffeine, piracetam. Part VI.

A group of 10 cats, both sexes, were studied for the effect of peroral administration of the meclophenoxate (Cetrexin, Léciva, 1.5 mg kg-1) + chlorprothixen (Chlorprothixen, Spofa, 0.045 mg kg-1) + caffeine (Coffeinum natrium benzoicum, Spofa, 0.15 mg kg-1) combination upon the fixation of conditioned alimentary motor reflexes to a sound signal in the course of a 10-week experiment. The mentioned combination of drugs demonstrated a beneficial protective influence on the fixed alimentary motor reflexes against laboratory stress. The results were compared with the earlier fixation of the same reflexes in another group of 11 cats under piracetam (Nootropil, U.C.B. 20 mg kg-1, s.c.). In both groups of animals, the development of reflexes was performed in regular alterations of experiments under the effect of the drugs and control experiments. The drugs were administered 1 hour before the experiments. Both groups of animals showed significantly fewer intersignal and other incorrect motor reactions on the days they were given the drugs than the controls did. The number of fixed correct reactions and their latencies displayed only moderate insignificant differences between the pharmacological trials and the controls. The conclusions is that the actual development of conditioned alimentary motor reflexes was not found to be influenced by the action of the mentioned drugs modifying psychological functions and mental states.