Conformational mimicry: Synthesis and solution conformation of a cyclic somatostatin hexapeptide containing a tetrazole cis amide bond surrogate

Potent, cyclic hexapeptide analogues of somatostatin are generally believed to adopt some common secondary structural features: a II′ β turn at one end of the cycle, and a type VI turn with a cis amide bond at the other. A proposed cis amide surrogate, the 1,5-disubstituted tetrazole, has been placed into a cyclic hexapeptide analog of somatostatin in order to constrain the putative cis amide bond. The final cyclization was done by either chemical or enzymatic means. The product, cyclo(Ala⁶-Tyr⁷-D -Trp⁸-Lys⁹-Val¹⁰-Phe¹¹-Ψ[CN₄]), was found to have 83% of the activity of somatostatin. Solution nmr analysis in DMSO/water revealed that the backbone as well as side chain χ₁ and χ₂ were well ordered. Relaxation matrix methods were used to extract distance restraints from the nuclear Overhauser effect spectroscopy data set, and these were used in a systematic search of torsional space to identify structures consistent with the nmr data. Restrained minimizations of these structures using a number of different force fields produced structures having the expected βII′ turn at D -Trp⁸-Lys⁹ and αβVIa turn in the Phe¹¹-Ψ[CN₄]-Ala⁶ portion of the molecule. The similarity of the minimized structures to those previously reported for cyclic hexapeptide analogues of somatostatin confirms the similarity of the tetrazole geometry to that of the cis amide in solution. © 1995 John Wiley & Sons, Inc.     

Aggregation of β-Amyloid Peptide Is Promoted by Membrane Phospholipid Metabolites Elevated in Alzheimer's Disease Brain

Abstract: Increased amounts of β-amyloid (Aβ) peptide deposits are found in Alzheimer's disease brain. These amyloid deposits have been implicated in the pathophysiology of this common dementing illness. Aβ peptides have been shown to be toxic to neurons in cell culture, and this toxicity is critically dependent on the aggregation of the peptide into cross-β-pleated sheet fibrils. Also, in vivo and postmortem NMR studies have shown changes in certain brain membrane phospholipid metabolites in normal aging and more extensive alterations in patients with Alzheimer's disease. The finding that membrane phospholipids affect the aggregation of Aβ suggests that the abnormalities in membrane metabolism found in Alzheimer's disease could affect the deposition of Aβ in vivo. Therefore, we examined the effect of membrane phospholipid metabolites that are altered in Alzheimer's disease brain on the aggregation of Aβ(1–40) using a light scattering method. Certain metabolites (glycerophosphocholine, glycerophosphoethanolamine, and α-glycerophosphate) augment the aggregation of Aβ. Other membrane phospholipid metabolites (phosphocholine, phosphoethanolamine, and inositol-1-phosphate) have no effect. We conclude that increased membrane phospholipid metabolite concentrations may play a role in the deposition of Aβ seen in normal aging and the even greater deposition of Aβ observed in Alzheimer's disease.     

Remote monitoring of parental incubation conditions in the greater sandhill crane

To monitor incubation conditions in nests of greater sandhill cranes, a radiotrans-mitting egg was built using six temperature sensors, a position sensor, and a light sensor. Sensor readings were received, along with time of observations, and stored in a computer. The egg was used to monitor incubation in nests of six pairs of cranes during 1987 and 1988. Ambient temperature was also measured. Analysis of covariance (ANCOVA) was used to relate highest egg temperature, core egg temperature, and lowest egg temperature to ambient temperature, time since the egg was last turned, and time since the beginning of incubation. Ambient temperature had the greatest effect on egg temperature (P < 0.0001), followed by the time since the beginning of incubation and time since the egg was last turned. Pair effect, the class variable in the ANCOVA, was also very significant (P < 0.0001). A nine-term Fourier series was used to estimate the average core egg temperature versus time of day and was found to fit the data well (r² = 0.94). The Fourier series will be used to run a mechanical incubator to simulate natural incubation conditions for cranes. © 1995 Wiley-Liss, Inc.     

Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco

cDNAs encoding three proteins from barley ( Hordeum vulgare ), a class-II chitinase (CHI), a class-II β-1,3-glucanase (GLU) and a Type-I ribosome-inactivating protein (RIP) were expressed in tobacco plants under the control of the CaMV 35S-promoter. High-level expression of the transferred genes was detected in the transgenic plants by Northern and Western blot analysis. The leader peptides in CHI and GLU led to accumulation of these proteins in the intercellular space of tobacco leaves. RIP, which is naturally deposited in the cytosol of barley endosperm cells, was expressed either in its original cytosolic form or fused to a plant secretion peptide (spRIP). Fungal infection assays revealed that expression of the individual genes in each case resulted in an increased protection against the soilborne fungal pathogen Rhizoctonia solani , which infects a range of plant species including tobacco. To create a situation similar to 'multi-gene' tolerance, which traditional breeding experience has shown to provide crops with a longer-lasting protection, several of these antifungal genes were combined and protection against fungal attack resulting from their co-expression in planta was evaluated. Transgenic tobacco lines were generated with tandemly arranged genes coding for RIP and CHI as well as GLU and CHI. The performance of tobacco plants co-expressing the barley transgenes GLU/ CHI or CHI/RIP in a Rhizoctonia solani infection assay revealed significantly enhanced protection against fungal attack when compared with the protection levels obtained with corresponding isogenic lines expressing a single barley transgene to a similar level. The data indicate synergistic protective interaction of the co-expressed anti-fungal proteins in vivo .     

Characteristics of the core protein of the aggregating proteoglycan from the Swarm rat chondrosarcoma

A ternary complex of hyaluronic acid-binding region and link protein bound to hyaluronic acid was isolated from limit clostripain digests of proteoglycan aggregates isolated from the Swarm rat chondrosarcoma. Under these conditions, the hyaluronic acid-binding region has a molecular weight of ? 65,000 (HA-BR₆₅). N-terminal amino acids in the complex were selectively ^l4C-carbamylated. The resulting derivatized HA-BR₆₅ was isolated, and tryptic peptide maps were prepared and developed on two-dimensional TLC sheets. A single, labeled peptide was obtained which gave a M_r by ? 8,000 by SDS-PAGE. Chymotrypsin digestion of the ternary complex reduced the molecular weight of HA-BR₆₅ to a polypeptide of ? 55,000 (HA-BR₅₅) which still retains the same N-terminal tryptic peptide. Partial digestion of proteoglycan aggregates with clostripain generated a series of larger intermediates with the hyaluronic acid-binding region. Direct SDS-PAGE analysis revealed one major intermediate with M_r ? 109,000 (HA-BR₁₀₉) as well as HA-BR₆₅. After chondroitinase digestion, two additional prominent intermediates were observed on a SDS-PAGE gel at M_r ? 120,000 (HA-BR₁₂₀) and ? 140,000 (HA-BR₁₄₀). All the intermediates were recognized by a monoclonal antibody specific for the hyaluronic acid-binding region, and all of them contained the same N-terminal tryptic peptide. The results indicate that the N terminus of the core protein is at the hyaluronic acid-binding end of the proteoglycan and that the chondroitin sulfate chains are first present on the core protein in a region between 109,000 and 120,000 molecular weight away from the N terminus.     

Stereoselective action of acylated crude papain toward mandelic and atrolactic hydrazides

Acylated crude papain has been shown to exert stereoselective behavior toward racemic hydrazides devoid of an amino acid residue, namely, (RS)-mandelic and (RS)-atrolactic hydrazides. These hydrazides functioned as nucleophiles to yield N¹,N²-diacylhydrazines. Several achiral acylating agents for the enzyme were chosen, including Z-glycine, BOC-glycine, AOC-glycine, and hippuric acid. With the exception of hippuric acid as the acylating agent, the reaction product, in every instance for these achiral hydrazides, consisted of an excess of the (+)-N¹,N²-diacylhydrazine. The relative rates of product formation for the mandelic hydrazides were considerably greater than for corresponding reactions with racemic atrolactic hydrazide. When chiral Z-l-alanine was employed to acylate crude papain, the stereoselective action was most pronounced, with the formation of a mixture of diastereoisomers consisting of 73% N¹-(Z-l-alanyl)-N²-[(R)-mandelyl]hydrazine. The relative reactivities for the electrophiles was Z-l-alanine ? Z-glycine ? hippuric acid ? AOC-glycine > BOC-glycine. The hydrazides of (R)-, (S)-mandelic, and (RS)-atrolactic acids were prepared by conversion of the corresponding acids to their esters by means of a catalytic dehydrating agent and subsequent treatment with a methanolic solution of hydrazine.     

Real-time ultrasonic imaging of the peripheral arteries: Technique, normal anatomy, and pathology

Real-time ultrasonic echo imaging of peripheral arteries promises to facilitate the management of selected patients with peripheral and extracranial arterial disease. This report outlines the technique of imaging the carotid system and portions of the arteries that supply the lower extremities. It also discusses the normal and pathologic anatomy of these arteries.     

Perception of the auxin signal at the plasma membrane of tobacco mesophyll protoplasts

Auxin-induced variations of transmembrane potential difference have been shown to be a useful tool for analyzing hormone sensitivity in tobacco protoplasts. Using this technique, we demonstrated that protoplasts derived from wild-type, an auxin-resistant mutant and Agrobacterium-rhizogenes transformed plants differed widely in the sensitivity of their electrical response to naphthalene acetic acid. We have used different antibodies, raised to auxin binding proteins (ABP) from maize coleoptiles, or to the axr1 gene product (ABP1), to test whether changes in auxin sensitivity can be correlated with the presence of tobacco proteins immunologically related to this ABP. Titrations indicated that 0.4 nM anti-ABP IgG inhibited 50% of the auxin-specific response of wild-type protoplasts, whereas 0.04 nM or 4 nM anti-ABP IgG were necessary to inhibit the response of mutant and transformed protoplasts, respectively, to the same extent. On wild-type protoplasts, blocking part of the immunoreactive sites with anti-ABP antibodies resulted in a decrease in auxin sensitivity of the electrical response (0.4 nM anti-ABP IgG inducing a 10–fold decrease), whereas addition of maize ABP increased this auxin sensitivity (1 pM ABP1 raised the sensitivity more than 1000–fold). The results obtained suggest that the auxin sensitivity detected by our assay system correlates with the amount of tobacco proteins immunologically related to the axr¹ gene product from maize. A hypothesis accounting for the presence of these proteins at the external surface of tobacco protoplasts and for the effects of hetero-logous maize ABP on auxin sensitivity is proposed.