Problems in the assessment of magnesium depletion in the rat by in vivo 31P NMR

Prior in vitro studies, utilizing 31P nuclear magnetic resonance (31P NMR) to measure the chemical shift (sigma) of beta-ATP and lengthening of the phosphocreatine spin-spin (T2) relaxation time, suggested an assessment of their efficacy in measuring magnesium depletion in vivo. Dietary magnesium depletion (Mg2+ decreases) produced markedly lower magnesium in plasma (0.44 vs 1.13 mmol/liter) and bone (130 vs 190 mumol/g) but much smaller changes in muscle (41 vs 45 mumol/g, P less than 0.01), heart (42.5 vs 44.6 mumol/g), and brain (30 vs 32 mumol/g). NMR experiments in anesthetized rats in a Bruker 7-T vertical bore magnet showed that in Mg2+ decreases rats there was a significant change in brain beta-ATP shift (16.15 vs 16.03 ppm, P less than 0.05). These chemical shifts gave a calculated free [Mg2+] of 0.71 mM (control) and 0.48 mM (Mg2+ decreases). In muscle the change in beta-ATP shift was not significant (Mg2+ decreases 15.99 ppm, controls 15.96 ppm), corresponding to a calculated free Mg2+ of 0.83 and 0.95 mM, respectively. Phosphocreatine T2 (Carr-Purcell, spin-echo pulse sequence) was no different with Mg2+ decreases in muscle in vivo (surface coil) (Mg2+ decreases 136, control 142 ms) or in isolated perfused hearts (Helmholtz coil) (control 83, Mg2+ decreases 92 ms). 31P NMR is severely limited in its ability to detect dietary magnesium depletion in vivo. Measurement of beta-ATP shift in brain may allow studies of the effects of interaction in group studies but does not allow prediction of an individual magnesium status.     

Timing, intensity, and duration of rehabilitation for hip fracture and stroke: report of a workshop at the National Center for Medical Rehabilitation Research

This article summarizes the proceedings of an NIH workshop on timing, intensity, and duration of rehabilitation for acute stroke and hip fracture. Participants concentrated on methodological issues facing investigators and suggested priorities for future research in this area.     

Cyclic MrIA: a stable and potent cyclic conotoxin with a novel topological fold that targets the norepinephrine transporter

Conotoxins, disulfide-rich peptides from the venom of cone snails, have created much excitement over recent years due to their potency and specificity for ion channels and their therapeutic potential. One recently identified conotoxin, MrIA, a 13-residue member of the chi-conotoxin family, inhibits the human norepinephrine transporter (NET) and has potential applications in the treatment of pain. In the current study, we show that the beta-hairpin structure of native MrIA is retained in a synthetic cyclic version, as is biological activity at the NET. Furthermore, the cyclic version has increased resistance to trypsin digestion relative to the native peptide, an intriguing result because the cleavage site for the trypsin is not close to the cyclization site. The use of peptides as drugs is generally hampered by susceptibility to proteolysis, and so, the increase in enzymatic stability against trypsin observed in the current study may be useful in improving the therapeutic potential of MrIA. Furthermore, the structure reported here for cyclic MrIA represents a new topology among a growing number of circular disulfide-rich peptides.     

Attention and successful episodic encoding: an event-related potential study

Event-related potentials (ERPs) were used to delineate the cerebral processes occurring when information is encoded into episodic memory and to determine how these processes are affected by divided attention. ERPs were recorded during encoding under focused or divided attention, and were selectively averaged on the basis of their retrieval during later free recall and recognition tests (with remember-know judgments). Items retrieved with conscious recollection of the encoding episode (remembered, recalled) were distinguished at encoding from later missed items by an enhanced left fronto-temporal negative wave (N340), a negative posterior sustained potential and a positive frontal sustained potential. These effects occurred independently of the level of attention. Items later retrieved on the basis of familiarity (known) elicited a larger N340 than missed items, but did not demonstrate the increased sustained potentials. We suggest that item-specific conceptual processing (N340) is sufficient to produce familiarity-based recognition, but additional elaborative processing (sustained interaction of frontal and posterior regions) is necessary for conscious recollection. The effect of divided attention on these processes was related to the difficulty of the secondary task, with the more difficult task causing greater and earlier interference.     

Ring flexibility within tricyclic antidepressant drugs

The internal flexibility of the central seven-membered ring of a series of tricyclic antidepressant drugs (TCAs), imipramine [1], amitriptyline [2], doxepin [3], and dothiepin [4], has been investigated by (1)H and (13)C nuclear magnetic (NMR) techniques. Two dynamic processes were examined: ring inversion and bridge flexing. (1)H NMR line-shape analysis was used to obtain ring inversion barriers for 2-4. These studies yielded energy barriers of 14.3, 16.7, and 15.7 +/- 0.6 kcal/mol for the hydrochloride salts of doxepin, dothiepin, and amitriptyline, respectively. The barriers for the corresponding free bases were lower by 0.6 kcal/mol on average. (13)C T(1) relaxation measurements were used to determine the degree of bridge flexing associated with the central seven-membered ring for all four compounds. By fitting the T(1) data to a two-state jump model, lifetimes and amplitudes of rapid bridge flexing motions were determined. The results show that imipramine has the fastest rate of bridge flexing, followed by amitriptyline, doxepin, and dothiepin. The pharmacological profiles of the TCAs are complex and they interact with many receptor sites, resulting in numerous side effects and a general lack of understanding of their precise mode of action in different anxiety-related disorders. They all have similar three-dimensional structures, which makes it difficult to rationalize their differing relative potency in different assays/clinical settings. However, the clear finding here that there are significantly different degrees of internal mobility suggests that molecular dynamics should be an additional factor considered when trying to understand the mode of action of this clinically important family of molecules.     

alpha-Conotoxins: nicotinic acetylcholine receptor antagonists as pharmacological tools and potential drug leads

Conotoxins are small disulfide rich peptides from the venoms of marine cone snails. They target specific nicotinic acetylcholine receptor (nAChR) subtypes with high affinity and potency and are therefore valuable as neuropharmacological probes and potential drug leads. This article gives a general overview of the chemical and biological features of alpha-conotoxins, including their pharmacology, binding interactions and structure. A detailed analysis of recently reported three-dimensional structures from members of different subfamilies of the alpha-conotoxins, including those with 3/5, 4/3, 4/6 and 4/7 spacings of their two intracysteine loops is given. The structures are generally well defined and represent useful frameworks for the display of amino acid residues to target molecules.     

Peptide--Fourth Australian Conference. From discovery to therapeutics--the new millennium

The Fourth Australian Peptide Conference continued the successful trend of the three previous conferences and encapsulated a range of peptide studies. As well as describing studies on the discovery, characterization and potential pharmaceutical applications of peptides, the meeting also featured a significant emphasis on new technologies, with proteomics and mass spectrometry having particular prominence. Several talks described peptides that have entered or are about to enter clinical trials. In his welcoming comments, the chair of the organizing committee, Milton Hearn (Monash University, Australia) noted that "peptide science and its attendant disciplines will play an ever increasing role in all fields of the life sciences and biotechnology". The breadth of disciplines represented at the conference reflected this increasing role.