The microtubule-binding fragment of microtubule-associated protein-2: location of the protease-accessible site and identification of an assembly-promoting peptide.

Thrombin cleavage of bovine brain microtubule-associated protein (MAP-2) yields two stable limit polypeptide fragments (28,000 and 240,000 Mr). The smaller cleavage product contains the microtubule-binding domain and is derived from the carboxyl terminus of MAP-2 while the 240,000 Mr fragment is derived from the amino terminus. The amino terminal sequence of the smaller cleavage product is homologous with the microtubule-binding fragment of tau in sequence and in a similar location relative to three imperfect octadecapeptide repeats implicated in microtubule binding. Peptides corresponding to the cleavage site and the three repeats of MAP-2 were synthesized. Only the second octadecapeptide repeat (VTSKCGSLKNIRHRPGGG) was capable of stimulating microtubule nucleation and elongation. Microtubules formed in the presence of this peptide displayed normal morphology and retained the inhibition properties of calcium ion, podophyllotoxin, and colchicine. Our result indicates that a region comprising only approximately 1% of the MAP-2 sequence can promote microtubule assembly.

Chemical cartography: finding the keys to the kinetic labyrinth.

Very high resolution lasers allow spectroscopic pictures to be taken following a collision between two molecular reactants. The features of these "pictures" are the electronic, vibrational, rotational, and translational motions of the atomic particles, which relate the quantum states of the reactants to the quantum states of the products. Such state-to-state kinetic information can be used to test the shape and nature of the interaction potential that controls the collision process. The potential itself is akin to a map of the terrain through mountains and valleys where elevation is a measure of energy instead of height. Accurate mapping of this potential surface leads to an understanding of the forces which control rates and mechanisms of chemical reactions. The application of four different advanced laser techniques to the study of collisions between "hot" hydrogen(H) atoms and carbon dioxide(CO(2)) molecules has provided a wealth of information about both reactive and nonreactive collisions for this system. The availability of data for rotationally, vibrationally, and translationally inelastic excitation of CO(2) by H atoms, when compared with data for reactive events producing OH + CO, provides insights into the dynamics of collisions between H and CO(2), and illustrates the future promise of these powerful techniques for elucidating features of potential energy surfaces.

Peptide binding and release by proteins implicated as catalysts of protein assembly.

Two members of the hsp70 family, termed hsc70 and BiP, have been implicated in promoting protein folding and assembly processes in the cytoplasm and the lumen of the endoplasmic reticulum, respectively. Short hydrophilic (8 to 25 residues) synthetic peptides have now been tested as possible mimics of polypeptide chain substrates to help define an enzymatic basis for these activities. Both BiP and hsc70 have specific peptide binding sites. Peptide binding elicits hydrolysis of adenosine triphosphate, with the subsequent release of bound peptide.

Noble gases in stratospheric dust particles: confirmation of extraterrestrial origin.

Noble gas elemental and isotopic ratios were measured in a group of 13 "chondritic" stratospheric dust particles. Neon and argon are present in "solar" proportions; xenon appears to be dominated by contributions from "planetary" sources. The apparent xenon concentration is higher than that measured in any bulk meteorite, approaching the concentration found in the noble gas-rich, acid-insoluble residues from carbonaceous chondrites.

An infrared spectral match between GEMS and interstellar grains.

Infrared spectral properties of silicate grains in interplanetary dust particles (IDPs) were compared with those of astronomical silicates. The approximately 10-micrometer silicon-oxygen stretch bands of IDPs containing enstatite (MgSiO3), forsterite (Mg2SiO4), and glass with embedded metal and sulfides (GEMS) exhibit fine structure and bandwidths similar to those of solar system comets and some pre-main sequence Herbig Ae/Be stars. Some GEMS exhibit a broad, featureless silicon-oxygen stretch band similar to those observed in interstellar molecular clouds and young stellar objects. These GEMS provide a spectral match to astronomical "amorphous" silicates, one of the fundamental building blocks from which the solar system is presumed to have formed.

Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels.

In cyclic nucleotide-gated channels (CNG), direct binding of cyclic nucleotides in the carboxy-terminal region is allosterically coupled to opening of the pore. A CNG1 channel pore was probed using site-directed cysteine substitution to elucidate conformational changes associated with channel opening. The effects of cysteine modification on permeation suggest a structural homology between CNG and KcsA pores. We found that intersubunit disulfide bonds form spontaneously between S399C residues in the helix bundle when channels are in the closed but not in the open state. While MTSET modification of pore-lining residues was state dependent, Ag(+) modification of V391C, in the inner vestibule, occurred at the same diffusion-limited rate in both open and closed states. Our results suggest that the helix bundle undergoes a conformational change associated with gating but is not the activation gate for CNG channels.

Molecular rearrangements in the ligand-binding domain of cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels are activated in response to the direct binding of cyclic nucleotides to an intracellular domain. This domain is thought to contain a beta roll and two alpha helices, designated the B and C helices. To probe the conformational changes occurring in the ligand-binding domain during channel activation, we used the substituted cysteine accessibility method (SCAM). We found that a residue in the beta roll, C505, is more accessible in unliganded channels than in liganded channels, whereas a residue in the C helix, G597C, is more accessible in closed channels than in open channels. These results support a molecular mechanism for channel activation in which the ligand initially binds to the beta roll, followed by an opening allosteric transition involving the relative movement of the C helix toward the beta roll.

Low temperature solution synthesis of silicon, germanium and Si-Ge axial heterostructures in nanorod and nanowire form.

Herein, we report the formation of silicon, germanium and more complex Si-SixGe1-x and Si-Ge axial 1D heterostructures, at low temperatures in solution. These nanorods/nanowires are grown using phenylated compounds of silicon and germanium as reagents, with precursor decomposition achieved at substantially reduced temperatures (200 °C for single crystal nanostructures and 300 °C for heterostructures), through the addition of a reducing agent. This low energy route for the production of these functional nanostructures as a wet chemical in high yield is attractive to meet the processing needs for next generation photovoltaics, batteries and electronics.

How do we compare with best practice? A completed audit of benzodiazepine and z-hypnotic prescribing.

OBJECTIVES: To compare benzodiazepine and z-hypnotic prescribing practices in an inpatient psychiatric unit to best practice standards. METHODS: Medication charts of all inpatients in the psychiatric unit, over a 1-week period, were reviewed. Details of current benzodiazepine and z-hypnotic prescriptions were collected. Information collected included the substance prescribed, duration and administration instructions. Feedback was communicated to medical practitioners through a presentation and email. A re-audit was completed 4 months later. RESULTS: There were increases in total benzodiazepine and z-hypnotic prescribing despite intervention. A reduction of 2 mg occurred in the mean regular dose of benzodiazepine prescribed. Lorazepam was the most prescribed benzodiazepine throughout. In both data sets, at least 50% of regular z-hypnotics and benzodiazepines were initiated before admission. There was an increase of 14% in regular benzodiazepines initiated in hospital exceeding 4 weeks in duration. In neither data collection did regular z-hypnotics initiated in hospital exceed this cut off. A greater number of individuals were in the process of being withdrawn from regular benzodiazepine or z-hypnotic prescriptions in the re-audit. There were minimal improvements in 'as required' prescribing as regards documentation of an indication, time limit and maximum dose. CONCLUSION: The increase in overall prescribing, despite intervention, maybe because these medications continued to be indicated in the acute presentations needing inpatient treatment. The small improvements in 'as required' prescribing patterns suggest that the intervention was limited in effecting change in this area.

Cer1p functions as a molecular chaperone in the endoplasmic reticulum of Saccharomyces cerevisiae.

Cer1p/Lhs1p/Ssi1p is a novel Hsp70-related protein that is important for the translocation of a subset of proteins into the yeast Saccharomyces cerevisiae endoplasmic reticulum. Cer1p has very limited amino acid identity to the hsp70 chaperone family in the N-terminal ATPase domain but lacks homology to the highly conserved hsp70 peptide binding domain. The role of Cer1p in protein folding and translocation was assessed. Deletion of CER1 slowed the folding of reduced pro-carboxypeptidase Y (pro-CPY) approximately twofold in yeast. In wild-type yeast under reducing conditions, pro-CPY can be found in a complex with Cer1p, while partially purified Cer1p is able to bind directly to peptides. Together, this suggests that Cer1p has a chaperoning activity required for proper refolding of denatured pro-CPY which is mediated by direct interaction with the unfolded polypeptide. Cer1p peptide binding and oligomerization could be disrupted by addition of ATP, confirming that Cer1p possesses a functional ATP binding site, much like Kar2p and other members of the hsp70 family. Interestingly, replacing the signal sequence of a CER1-dependent protein with that of a CER1-independent protein did not relieve the requirement of CER1 for import. This result suggests that an interaction with the mature portion of the protein also is important for the translocation role of Cer1p. The CER1 RNA levels increase at lower temperatures. In addition, the effects of deletion on folding and translocation are more severe at lower temperatures. Therefore, these results suggest that Cer1p provides an additional chaperoning activity in processes known to require Kar2p. However, there appears to be a greater requirement for Cer1p chaperone activity at lower temperatures.

High-resolution solution structure of the 18 kDa substrate-binding domain of the mammalian chaperone protein Hsc70.

The three-dimensional structure for the substrate-binding domain of the mammalian chaperone protein Hsc70 of the 70 kDa heat shock class (HSP70) is presented. This domain includes residues 383-540 (18 kDa) and is necessary for the binding of the chaperone with substrate proteins and peptides. The high-resolution NMR solution structure is based on 4150 experimental distance constraints leading to an average root-mean-square precision of 0.38 A for the backbone atoms and 0.76 A for all atoms in the beta-sandwich sub-domain. The protein is observed to bind residue Leu539 in its hydrophobic substrate-binding groove by intramolecular interaction. The position of a helical latch differs dramatically from what is observed in the crystal and solution structures of the homologous prokaryotic chaperone DnaK. In the Hsc70 structure, the helix lies in a hydrophobic groove and is anchored by a buried salt-bridge. Residues involved in this salt-bridge appear to be important for the allosteric functioning of the protein. A mechanism for interdomain allosteric modulation of substrate-binding is proposed. It involves large-scale movements of the helical domain, redefining the location of the hinge area that enables such motions.

Conformational change of chaperone Hsc70 upon binding to a decapeptide: a circular dichroism study.

The conformation of bovine Hsc70, a 70-kDa heat shock cognate protein, and its conformational change upon binding to decapeptides, was studied by CD spectroscopy and secondary structure prediction (Chou, P.Y. & Fasman, G.D., 1974, Biochemistry 13, 222-245). The CD spectra were analyzed by the LINCOMB method, as well as by the convex constraint analysis (CCA) method (Perczel, A., Park, K., & Fasman, G.D., 1992, Anal. Biochem. 203, 83-93). The result of the CD analysis of Hsc70 (15% alpha-helix, 24% beta-sheet, 24% beta-turn, and 38% remainder) was very similar to the predicted secondary structure for the beta-sheet (24%) and the beta-turn (29%). However, there is disagreement between the alpha-helical content by CD analysis (15%) and the predicted structure (30%). In spite of the fact that the decapeptides contained a considerable amount of beta-sheet (22%), the interaction of the heat shock protein with the peptide resulted in an overall decrease in the content of beta-sheet conformation (-15%) of the complex. This may be due to induction of a molten globule state. The result of the CCA analysis indicated that the Hsc70 undergoes a conformational change upon binding the decapeptides.

Topology and dynamics of the 10 kDa C-terminal domain of DnaK in solution.

Hsp70 molecular chaperones contain three distinct structural domains, a 44 kDa N-terminal ATPase domain, a 17 kDa peptide-binding domain, and a 10 kDa C-terminal domain. The ATPase and peptide binding domains are conserved in sequence and are functionally well characterized. The function of the 10 kDa variable C-terminal domain is less well understood. We have characterized the secondary structure and dynamics of the C-terminal domain from the Escherichia coli Hsp70, DnaK, in solution by high-resolution NMR. The domain was shown to be comprised of a rigid structure consisting of four helices and a flexible C-terminal subdomain of approximately 33 amino acids. The mobility of the flexible region is maintained in the context of the full-length protein and does not appear to be modulated by the nucleotide state. The flexibility of this region appears to be a conserved feature of Hsp70 architecture and may have important functional implications. We also developed a method to analyze 15N nuclear spin relaxation data, which allows us to extract amide bond vector directions relative to a unique diffusion axis. The extracted angles and rotational correlation times indicate that the helices form an elongated, bundle-like structure in solution.

Enhancement of hydrocortisone permeation of human and hairless mouse skin by 1-dodecylazacycloheptan-2-one.

The influence of 1-dodecylazacycloheptan-2-one (Azone) on the in vitro permeation of hairless mouse skin and human epidermis by hydrocortisone was studied as a function of the amount of Azone solubilized and/or emulsified into aqueous media applied to the membranes using the infinite-dose technique. The permeability-enhancing effect of Azone increases with increasing Azone total concentrations until 0.1% is reached with mouse skin and 0.01% is reached for human epidermis. Thereafter, permeabilities for both tissues drop systematically. The maximally enhanced permeability in mouse skin approached that for mouse skin stripped of its stratum corneum. The peak permeability in human epidermis is an order of magnitude smaller than for mouse skin with the duration of Azone treatment required to achieve the full effect in human epidermis being twice that for mouse skin (approximately 20 h vs approximately 12 h). Thus, there is a profound difference in Azone's action on these two tissue types. It was also established that the affinity of an enhancer for a permeant drug can significantly offset its ability to enhance permeability. Specifically, hydrocortisone was found to partition significantly into the Azone-rich phase of the emulsion, lowering its concentration (and its thermodynamic activity) in the continuous aqueous phase and thereby reducing its flux through the skin. This physiochemical effect was profound enough to nullify the intrinsic permeability-enhancing effect of Azone as the total Azone concentration was raised to 10%.

Physicochemical characterization of the human nail: I. Pressure sealed apparatus for measuring nail plate permeabilities.

Diffusion characteristics of the nail plate are necessary in providing the baselines for rational topical management of nail infections. In order to develop such baselines a unique stainless steel diffusion cell has been designed. The cell permits the exposure of 0.38 cm2 of nail plate to a bathing medium which is stirred by small motors mounted above the cell. The diffusion of water, methanol and ethanol at constant temperature (37 degrees C), has been examined over periods up to 4 h. Average permeability coefficients of water, methanol and ethanol were determined as 16.5 +/- 5.9 X 10(-3) cm hr-1, 5.6 X 10(-3) cm hr-1 and 5.8 +/- 3.1 X 10(-3) cm hr-1 respectively. Moreover rates of diffusion across the nail were inversely proportional to nail thickness. Based on methanol data, nail plate barrier property appears stable for long periods of aqueous immersion.

Some general influences of n-decylmethyl sulfoxide on the permeation of drugs across hairless mouse skin.

The influences of n-decylmethyl sulfoxide (NDMS) on the permeation of phenyl propanolamine, propranolol, acyclovir, and hydrocortisone through hairless mouse skin were investigated. Permeation of these compounds increased systematically with increasing NDMS concentration up to a limiting value for the permeability coefficient of about 0.1 cm/h. Permeability coefficients obtained with stripped skin indicated that, where NDMS has its maximal effect, the compounds diffuse through the skin as if there were no stratum corneum. Diffusion lag times of the test compounds through skin sections exposed to NDMS were far shorter than lag times in control experiments. The kinetics of NDMS action were assessed by pretreating skin sections for various periods of time with the enhancer and then assessing permeability. When applied as a 10 mM aqueous solution, the full effect of NDMS was obtained in 3 h of exposure and the effect lasted for at least 24 h. Treating the skin with ethanol/water (2:1) to elute NDMS after the pretreatment did not diminish its effect. Propranolol and phenyl propanolamine solubilized NDMS, presumably through the formation of mixed micelles, which had a profound effect on the permeation rates of both drug and enhancer.

Physicochemical study of percutaneous absorption enhancement by dimethyl sulfoxide: dimethyl sulfoxide mediation of vidarabine (ara-A) permeation of hairless mouse skin.

Dimethyl sulfoxide's (DMSO) concentration-dependent influences on its own permeation rate through hairless mouse skin and on the concurrent permeation rates of water and the antiviral drug vidarabine (ara-A) have been studied at 37 degrees C using in vitro diffusion cells. Solubilities of ara-A in DMSO-water mixtures were also determined in order to assess ara-A's relative thermodynamic activity in the binary solvent media used in the mass transfer studies. Solubilities increased exponentially with increasing percentages of DMSO. Activity coefficients decreased accordingly. When the same DMSO medium was placed in each side of diffusion cell (balanced solvent configuration) permeability coefficients for ara-A decreased exactly as ara-A's solubility increased up to a 50% DMSO concentration, indicating the observed decreases in the mass transfer coefficients have thermodynamic origins. When DMSO media were placed in either the donor or receiver side of the cell up to the same 50% concentration point and opposed by a normal saline medium on the other side (asymmetric solvent configurations), the permeability of ara-A did not decrease and at some DMSO levels was substantially increased, behavior in marked departure from thermodynamic control. The behavior disparity between the 2 configurations of the cell suggests that cross-currents of solvents play a role in permeability enhancement. Regardless of solvent configuration, permeability coefficients for ara-A at 90 and 100% DMSO strengths were exaggeratedly large, consistent with severe impairment of the stratum corneum. Similar overall permeability behavior was observed for the 2 solvents, water and DMSO. Possible underlying mechanisms for these effects and the relative importance of the various mechanisms of DMSO enhancement as a function of DMSO's concentration and configuration are discussed.

Permeability of thermally damaged skin: I. Immediate influences of 60 degrees C scalding on hairless mouse skin.

Freshly sacrificed hairless mice were burned dorsally by direct contact with 60 degrees C water for periods ranging from 15 seconds to 8 min. Wounds ranging in degree from superficial epidermis damage to injury penetrating well into subcutaneous musculature were inflicted. Burned skin sections and reference abdominal skin sections were excised, placed in diffusion cells and investigated with regards to their permeabilities to water, methanol, ethanol, n-butanol and n-octanol. The data were couched in terms of ratios of permeability coefficients of burned skin to normal skin (scalding coefficients) for the same animal. Scalding increased permeability of skin to all compounds studied but the effects leveled out by 60 seconds. Protracted scalding was without great effect despite progressively increased depth of damage to the tissue as noted in histological sections. The degree of lost barrier competency attributable to 60 degrees C scalding was not marked for any compound but was definitely different for different alkanols. An approximately 3-fold permeability increase was noted with n-butanol, the most affected compound. The data demonstrate that near instantaneous alterations in permeability of skin accompany scalding, that decreased barrier competency does not correlate with the severity of a burn as measured in depth of the burn, and that thermal alteration of permeabilities is dependent on the physicochemical characteristics of the permeants.

Hydration and percutaneous absorption: I. Influence of hydration on alkanol permeation through hairless mouse skin.

A method to study the influence of hydration on skin permeability where the skin is immersed in saline for up to 30 hr and under circumstances where a steady state rate of permeation can be established in several minutes is indicated. These circumstances allow multiple, sequential runs over a period where the permeability coefficients of some chemicals are gradually changing. It has been found that the permeabilities of water, methanol and ethanol are little affected by such hydration. However, there is a doubling of the permeability coefficients of butanol and hexanol during the first 10 hr of immersion. More hydrophobic alkanols seem to be less sensitive to the protracted aqueous conditioning. In general the results indicate that there are complex molecular structure-permeability relationships operating in skin. More specifically, the hydration effects are insightful with respect to developing barrier models for skin as they are further indications that different parallel diffusional paths are followed by polar and semi- and nonpolar species.

In vitro staining of islets of Langerhans for fluorescence-activated cell sorting.

A previously described technique from the author's laboratories for purification of pancreatic islets by fluorescence-activated cell sorting used the dye neutral red (NR) to obtain specific fluorescence of islets sufficient to give a sorting signal. A major drawback with this technique was the need to inject the dye intravascularly before excision of the pancreas. Preliminary investigations showed that NR would produce selective staining of islets by topical application in vitro but only at low concentrations that were insufficient to give fluorescence strong enough for sorting. The chelating agent dithizone (DTZ) produces bright red staining of islets by topical application in vitro. Further studies showed that dithizone-stained islets exhibited moderately strong fluorescence that faded too quickly for reliable sorting. By combining both NR and DTZ staining in vitro, selective fluorescence of islets was obtained that was sufficient to allow efficient sorting. Using the combined DTZ/NR stain the yield of islets obtained by sorting from a single rat pancreas was 569 +/- 72 (n = 16), corresponding to 83% of the islets present in the digest. The mean purity of the preparation, confirmed by histologic examination, was 80%. The viability of the islets was shown to be good both by supravital staining and by the successful correction of streptozotocin diabetes in syngeneic rats following transplantation of sorted islets.