Structural analysis of the neuronal SNARE protein syntaxin-1A.

Intracellular trafficking depends on the docking and fusion of transport vesicles with cellular membranes. Central to docking and fusion is the pairing of SNARE proteins (soluble NSF attachment protein receptors) associated with the vesicle and target membranes (v- and t-SNAREs, respectively). Here, the X-ray structure of an N-terminal conserved domain of the neuronal t-SNARE syntaxin-1A was determined to a resolution of 1.9 A using multiwavelength anomalous diffraction. This X-ray structure, which is in general agreement with an NMR structure of a similar fragment, provides new insight into the interaction surface between the N-terminal domain and the remainder of the protein. In vitro characterization of the intact cytoplasmic domain of syntaxin revealed that it forms dimers, and probably tetramers, at low micromolar concentrations, with concomitant structural changes that can be detected by limited proteolysis. These observations suggest that the promiscuity characteristic of pairing between v-SNAREs and t-SNAREs extends to the formation of homo-oligomeric t-SNARE complexes as well. They also suggest a potential role for the neuronal Sec1 protein (nSec1) in preventing the formation of syntaxin multimers.

Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity.

The antimicrobial peptide LL-37 belongs to the cathelicidin family and is the first amphipathic alpha-helical peptide isolated from human. LL-37 is considered to play an important role in the first line of defence against local infection and systemic invasion of pathogens at sites of inflammation and wounds. Understanding its mode of action may assist in the development of antimicrobial agents mimicking those of the human immune system. In vitro studies revealed that LL-37 is cytotoxic to both bacterial and normal eukaryotic cells. To gain insight into the mechanism of its non-cell-selective cytotoxicity, we synthesized and structurally and functionally characterized LL-37, its N-terminal truncated form FF-33, and their fluorescent derivatives (which retained structure and activity). The results showed several differences, between LL-37 and other native antimicrobial peptides, that may shed light on its in vivo activities. Most interestingly, LL-37 exists in equilibrium between monomers and oligomers in solution at very low concentrations. Also, it is significantly resistant to proteolytic degradation in solution, and when bound to both zwitterionic (mimicking mammalian membranes) and negatively charged membranes (mimicking bacterial membranes). The results also showed a role for the N-terminus in proteolytic resistance and haemolytic activity, but not in antimicrobial activity. The LL-37 mode of action with negatively charged membranes suggests a detergent-like effect via a 'carpet-like' mechanism. However, the ability of LL-37 to oligomerize in zwitterionic membranes might suggest the formation of a transmembrane pore in normal eukaryotic cells. To examine this possibility we used polarized attenuated total reflectance Fourier-transform infrared spectroscopy and found that the peptide is predominantly alpha-helical and oriented nearly parallel with the surface of zwitterionic-lipid membranes. This result does not support the channel-forming hypothesis, but rather it supports the detergent-like effect.

Doxepin's effects on chronic pain and depression: a controlled study.

Sixty patients with chronic pain of the low back or cervical spine concomitant with clinical depression were studied in a 6-week, randomized, double-blind comparison of doxepin and placebo. Significant improvements in the doxepin-treated group compared to placebo or to baseline values were seen on Hamilton depression scores, Global Assessment Scale scores, pain severity, percent of time pain felt, and effect of pain on activity, sleep, and muscle tension. Some improvements were observed after 1 week of treatment; the most improvement occurred at 6 weeks, when the mean doxepin dosage was approximately 200 mg/day and plasma doxepin and nordoxepin averaged 80 ng/ml. No significant harmful effects were observed. Neither plasma beta-endorphin nor enkephalin-like activity demonstrated significant differences from baseline. These data indicate that doxepin is a valuable treatment for patients with chronic pain and depression.

Variation in the carbon isotope composition of a plant with crassulacean Acid metabolism.

The content of (13)C varies in plants with Crassulacean acid metabolism. Differences up to 3.5 per thousand in the (13)C/(12)C ratios were observed between leaves of different age in the same plant of Bryophyllum daigremontianum. Soluble and insoluble carbon in the same leaf differed up to 8 per thousand, the largest difference occurring in the leaves with the highest Crassulacean acid metabolism activity. Models to account for the isotope discrimination by C(3), C(4), and Crassulacean acid metabolism plants are proposed.

Carbon fixation and isotope discrimination by a crassulacean plant: dependence on the photoperiod.

Variations of more than 1 percent are observed in the carbon-13 to carbon-12 ratio of extracts of leaves of the succulent Kalanchoe blossfeldiana when the photoperiod is changed from long to short days. This indicates that the mechanism of carbon fixation switches from the Calvin (C(3)) pathway to the Hatch-Slack (C(4)) pathway of primary enzymic operation. The variations observed in the isotope compositions are tentatively explained by a model.

Carbon-14 in patagonian tree rings.

The radiocarbon activity found in tree rings from southern Argentina shows secular fluctuations which are synchronous with and of the same amplitude as those known for the Northern Hemisphere. Comparable measurements indicate that the activity in Patagonian trees is about five per mil lower than in European trees.