Local Conformation and Dynamics of Isoleucine in the Collagenase Cleavage Site Provide a Recognition Signal for Matrix Metalloproteinases

The mechanism by which enzymes recognize the “uniform” collagen triple helix is not well understood. Matrix metalloproteinases (MMPs) cleave collagen after the Gly residue of the triplet sequence Gly∼[Ile/Leu]-[Ala/Leu] at a single, unique, position along the peptide chain. Sequence analysis of types I-III collagen has revealed a 5-triplet sequence pattern in which the natural cleavage triplets are always flanked by a specific distribution of imino acids. NMR and MMP kinetic studies of a series of homotrimer peptides that model type III collagen have been performed to correlate conformation and dynamics at, and near, the cleavage site to collagenolytic activity. A peptide that models the natural cleavage site is significantly more active than a peptide that models a potential but non-cleavable site just 2-triplets away and NMR studies show clearly that the Ile in the leading chain of the cleavage peptide is more exposed to solvent and less locally stable than the Ile in the middle and lagging chains. We propose that the unique local instability of Ile at the cleavage site in part arises from the placement of the conserved Pro at the P₃ subsite. NMR studies of peptides with Pro substitutions indicate that the local dynamics of the three chains are directly modulated by their proximity to Pro. Correlation of peptide activity to NMR data shows that a single locally unstable chain at the cleavage site, rather than two or three labile chains, is more favorable for cleavage by MMP-1 and may be the determining factor for collagen recognition.     

Die Karpelle vonEranthis hiemalis undCimicifuga americana als weitere Verbindungsglieder zwischen peltaten und epeltaten Karpellen

Zusammenfassung Die Karpelle vonEranthis hiemalis undCimicifuga americana vermitteln den Übergang zwischen den peltaten und den epeltaten Karpellen, da ihre Querzone nicht mehr wie bei den peltaten Karpellen im Spreitenbereich liegt und noch nicht wie bei den epeltaten Karpellen gänzlich unterdrückt ist, sondern sich intermediär verhält, nämlich in den Karpellstiel verlagert ist. Die bei ihnen also auffallend tiefe Lage der Querzone erweist sich als eine Folge der späten Querzonenanlegung.Ähnliche Verhältnisse wurden bisher nur vonGrevillea Thelemanniana bekannt.     

Modeling the dynamics of choice

A simple linear-operator model both describes and predicts the dynamics of choice that may underlie the matching relation. We measured inter-food choice within components of a schedule that presented seven different pairs of concurrent variable-interval schedules for 12 food deliveries each with no signals indicating which pair was in force. This measure of local choice was accurately described and predicted as obtained reinforcer sequences shifted it to favor one alternative or the other. The effect of a changeover delay was reflected in one parameter, the asymptote, whereas the effect of a difference in overall rate of food delivery was reflected in the other parameter, rate of approach to the asymptote. The model takes choice as a primary dependent variable, not derived by comparison between alternatives—an approach that agrees with the molar view of behaviour.     

Trypanosoma cruzi: inhibition by spirogermanium hydrochloride

Spirogermanium is an antineoplastic agent that has been shown to be useful for the treatment of a variety of solid tumors and Plasmodium falciparum infection. We found that this agent, at concentrations of 1-10 micrograms/ml, markedly inhibited the growth of epimastigotes of Trypanosoma cruzi. This inhibition of growth was seen in liver infusion tryptose cultures as well as on agar where colonial growth was inhibited markedly. Ultrastructural studies demonstrated that affected organisms were round and swollen and contained vacuoles, lamellar structures, and multivesicular bodies. Spirogermanium also significantly decreased the growth of intracellular amastigotes in myotubes. Pretreatment of myotubes with the agent protected them from infection with trypomastigotes but tachyzoites of Toxoplasma sp. readily infected pretreated cells. These data suggest that spirogermanium may be useful as a chemotherapeutic agent against T. cruzi.     

The specificity of thiourea, dimethylthiourea and dimethyl sulphoxide as scavengers of hydroxyl radicals. Their protection of alpha 1-antiproteinase against inactivation by hypochlorous acid.

Thiourea and dimethylthiourea are powerful scavengers of hydroxyl radicals (.OH), and dimethylthiourea has been used to test the involvement of .OH in several animal models of human disease. It is shown that both thiourea and dimethylthiourea are scavengers of HOCl, a powerful oxidant produced by neutrophil myeloperoxidase. Hence the ability of dimethylthiourea to protect against neutrophil-mediated tissue damage cannot be used as evidence for a role of .OH in causing such damage. Dimethyl sulphoxide also reacts with HOCl, but at a rate that is probably too low to be biologically significant at dimethyl sulphoxide concentrations up to 10 mM. Neither mannitol nor desferrioxamine, at the concentrations normally used in radical-generating systems, appears to react with HOCl.     

Evidence that ATP-dependent Ca2+ transport in rat parotid microsomal membranes requires charge compensation.

ATP-dependent Ca2+ transport was investigated in a rat parotid microsomal-membrane preparation enriched in endoplasmic reticulum. Ca2+ uptake, in KCl medium, was rapid, linear with time up to 20 s, and unaffected by the mitochondrial inhibitors NaN3 and oligomycin. This Ca2+ uptake followed Michaelis-Menten kinetics, and was of high affinity (Km approximately 38 nM) and high capacity (approximately 30 nmol/min per mg of protein). In the presence of oxalate, Ca2+ uptake continued to increase for at least 5 min, reaching an intravesicular accumulation approx. 10 times higher than without oxalate. Ca2+ uptake was dependent on univalent cations in the order K+ = Na+ greater than trimethylammonium+ greater than mannitol and univalent anions in the order Cl- greater than acetate- greater than Br- = gluconate- = NO3- greater than SCN-. Ca2+ uptake was not elevated if membranes were incubated in the presence of a lipophilic anion (NO3-) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Ca2+ transport was altered by changes in the K+-diffusion potential of the membranes. A relatively negative K+-diffusion potential increased the initial rate of Ca2+ accumulation, whereas a relatively positive potential decreased Ca2+ accumulation. In the presence of an outwardly directed K+ gradient, nigericin had no effect on Ca2+ uptake. In aggregate, these studies suggest that the ATP-dependent Ca2+-transport mechanism present in rat parotid microsomal membranes exhibits an electrogenic Ca2+ flux which requires the movement of other ions for charge compensation.     

Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.     

Calcium entry in rat parotid acinar cells

Conclusions While it is generally accepted that Ca²⁺ plays an important regulatory role in the physiology of a number of non-excitable cells, the mechanisms which regulate intracellular [Ca²⁺ are far from well established. Ca²⁺ transporting mechanisms which distribute Ca²⁺ intracellularly as well as those which allow influx of extracellular Ca²⁺ are involved in mediating intracellular Ca²⁺ homestasis. In this paper we have described recent studies on the regulation of the Ca²⁺ influx system in the data, it appears that the process of Ca²⁺ entry is extremely complex and may involve several levels of regulation. Understanding the molecular basis of these regulatory mechanisms presents a challeging problem for future studies.     

The 5S DNA units of bread wheat (Triticum aestivum)

A collection of 44 cloned 5S DNA units fromTriticum aestivum cv. Chinese Spring were grouped into 12 sequence-types based on sequence similarity and the respective consensus sequences were then produced. The relationship between these 12 consensus sequences (T. aestivum S 1-S 8 andT. aestivum L 1-L 4), together with two clones sequenced byGerlach andDyer, and the 5S DNA consensus sequences from diploidTriticum spp. were then determined by numerical methods. Both phenetic and cladistic analyses were carried out. The following wheat 5S DNA sequences were found to group with respective sequences from diploidTriticum spp.:T. aestivum S 4, S 6 withT. tauschii S;T. aestivum S 3 withT. monococcum S andT. monococcum S-Rus 7;T. aestivum L 1 andT. aestivum L-G&D withT. speltoides L;T. aestivum L 2, L 3 withT. tauschii L;T. aestivum L 4 withT. monococcum L andT. monococcum L-Rus 12. The analyses suggested that 5 out of the 65S Dna loci present in wheat were identified at the sequence level. The locus that could not be identified in this analysis was the5S Dna-B 1 locus. A group ofT. aestivum sequences (T. aestivum S 1, S 7, S 8, S-G&D) were found to be distinct from the other 5S DNA sequences in the data base. The existence of the distinct group of 5S DNA sequences suggests that there is a gap in our current understanding of wheat evolution with respect to the5S Dna loci.