Refolding the Engrailed Homeodomain: Structural Basis for the Accumulation of a Folding Intermediate

The ultrafast folding pathway of the engrailed homeodomain has been exceptionally well characterized by experiment and simulation. Helices II and III of the three-helix bundle protein form the native helix-turn-helix motif as an on-pathway intermediate within a few microseconds. The slow step is then the proper docking of the helices in ∼15 μs. However, there is still the unexplained puzzle of why helix docking is relatively slow, which is part of the more general question as to why rearrangements of intermediates occur slowly. To address this problem, we performed 46 all-atom molecular dynamics refolding simulations in explicit water, for a total of 15 μs of simulation time. The simulations started from an intermediate state structure that was generated in an unfolding simulation at 498 K and was then quenched to folding-permissive temperatures. The protein refolded successfully in only one of the 46 simulations, and in that case the refolding pathway mirrored the unfolding pathway at high temperature. In the 45 simulations in which the protein did not fully fold, nonnative salt bridges trapped the protein, which explains why the protein folds relatively slowly from the intermediate state.     

Incorporation of an enrichment program into a study protocol involving long-term restraint in macaques

Nonhuman primates might experience stress during periods of restraint associated with research procedures. In an attempt to minimize such stress, the authors describe an enrichment program they designed for use with restrained adult male rhesus macaques.     

Specific glycanforms of type IX collagen accumulate in embryonic chick sterna after 17 days of development

Type IX collagen is a key component of the extracellular matrix of cartilage where it occurs at the surfaces of type II collagen fibrils as a glycanated molecule. The function of the glycosaminoglycan (GAG) side chain of the molecule is, however, unknown. We have shown that type IX collagen in chicken sternal cartilage is synthesized with a unimodal distribution of GAG chain size, but at post 17 days of development three predominant glycanforms of type IX collagen accumulate. Such accumulation did not occur in sterna from day 15 embryos. In day 17 embryos predominant glycanforms were found in the caudal region of the sternum. By day 19 of development the three predominant glycanforms are widespread throughout the caudal and cephalic regions. The results indicate that developmental and anatomical changes occur to type IX collagen that depend on the size of the GAG chain attached to the alpha2(IX) chain of the molecule.      

Plant and bacterial mucilages of the maize rhizosphere: Comparison of their soil binding properties and histochemistry in a model system

Mucilages from the root tips of axenically-grown maize and from a bacterium (Cytophaga sp.) isolated from the rhizosheaths of field-grown roots, were immobilized by drying onto nylon blotting membrane. The mucilage plaques remained in place through repeated rewettings and histochemical treatments. Staining of the plaques showed that both mucilages included acidic groups, and 1,2 diols (the latter notably fewer in bacterial mucilage). Bacterial mucilage plaques stained strongly for protein, plant mucilage was unstained. Plaques of both mucilages bound soil particles strongly if soil was applied to wet mucilage and then dried. Bound soil was not lost with rewetting. Dry weight and densitometer measurements showed that bacterial mucilage bound about 10% more soil than the same surface area of root-cap mucilage. Pretreatment of plaques with periodate oxidation eliminated most soil binding by root-cap mucilage but this was completely reversible by reduction with borohydride. Soil binding to bacterial mucilage was unaffected by periodate but much diminished by borohydride pretreatment (partially restored by subsequent oxidation). Neither pretreatment with cationic dyes nor preincubation in pectinase, pectin methylesterase or protease affected subsequent soil binding by the mucilage plaques. Pretreatment of root-cap mucilage plaques with lectins specific for component sugars also did not alter soil binding. It is concluded that mucilages of both plant and bacterial origin can contribute to the adhesion and cohesion of maize rhizosheaths, but each by a different mechanism. Binding by root-cap mucilage depends on 1,2 diol groups of component sugars, that of bacterial mucilage does not, and is likely to be protein mediated. ei]Section editor: R O D Dixon     

Mutations affecting lipoamide dehydrogenases of Pseudomonas putida.

Pseudomonas putida grown on valine produces two lipoamide dehydrogenases, LPD-glu (Mr, 56,000 and LPD-val (Mr, 49,000). The 49,000-dalton protein is used by P. putida for branched-chain keto acid dehydrogenase, whereas the 56,000-dalton protein is presumably used for pyruvate and 2-ketoglutarate dehydrogenases. The objective of this study was to isolate and characterize mutants of P. putida with mutations affecting lipoamide dehydrogenases in order to study the relationship of these two proteins. Mutant JS287 lacked LPD-val, the lipoamide dehydrogenase which is induced by growth on valine and is specific for branched-chain keto acid dehydrogenase, and had normal amounts of LPD-glu, the lipoamide dehydrogenase which is formed during growth on glucose and which is probably used by both pyruvate and 2-ketoglutarate dehydrogenases. Mutant JS94 was a pleiotropic mutant with defects in 2-ketoglutarate, branched-chain, and lipoamide dehydrogenases. Proteolysis of LPD-glu and LPD-val produced completely different digestion products, suggesting that these two proteins are products of separate structural genes. Antisera prepared against LPD-glu reacted only with LPD-glu, whereas antisera prepared against LPD-val reacted with LPD-val and cross-reacted with LPD-glu. Although mutant JS94 did not produce active lipoamide dehydrogenase, cell-free extracts of this mutant contained a protein which cross-reacted with anti-LPD-val.     

Reprogramming of cells following wounding in pea (Pisum sativum L.) roots. II. The effects of caffeine and colchicine on the development of new vascular elements

Summary Interference with the normal progression of the cell cycle by the drugs caffeine and colchicine does not prevent parenchyma cells in the cortex of pea roots from being reprogrammed to become tracheary and sieve elements following severance of the vascular cylinder of the root. The pattern of secondary wall deposition of the newly differentiated tracheary elements is highly aberrant in the presence of colchicine but is of normal appearance in the caffeinetreated roots. In each case, the new sieve elements have sieve plates and lateral sieve areas with callose deposits. Induction and redifferentiation are achieved in the absence of cell division and microtubules in colchicine-treated roots.Bi- and multi-nucleate cells are produced by both drugs. Microtubules are still present in the caffeine-treated roots but cell plate formation is inhibited. The partitioning of the multinucleate cortical cells by the interdigitation of free-growing walls between the nuclei occurs in the presence of caffeine but not colchicine.     

ION FLUXES AND MODIFICATION OF THE EXTRACELLULAR MATRIX DURING GAMETE RELEASE IN FUCOID ALGAE

A rapid phase transition in the extracellular matrix (ECM) of the reproductive tissues (= receptacles) has been proposed to cause gamete release in fucoid algae. We tested this model with cryoanalytical energy dispersive x-ray microanalysis (EDX) scanning electron microscopy (SEM) of rapidly frozen hydrated receptacles of Silvetia compressa Serrão, Cho, Boo et Brawley that were planed to smooth faces using a cryoultramicrotome. Every receptacle typically contained a region(s) of intracellular accumulation of K and Cl and a region(s) of efflux of K and Cl to the ECMs, regardless of treatment (e.g. calm vs. shaken conditions in the light, anthracene-9-carboxylic acid). Although longitudinal variations in [K] and [Cl] between ECM and cells were common, a tissue within any transverse plane of the receptacle was in the same state of efflux or accumulation of K and Cl. The strongest and most extensive effluxes occurred during the time of irreversible commitment to gamete release (i.e. 2–4 min darkness after light potentiation). A receptacle may oscillate rapidly between global states of accumulation and efflux at this time. The polysaccharide collar between the oogonium and stalk cell expanded during Cl and K efflux into the ECM of the conceptacle and condensed when [K] and [Cl] were low in the conceptacle ECM. During periods of strong efflux associated with gamete release, the collar and other components of the exochiton ruptured. Detached gametangia were guided to the pore by the paraphyses. The ECM of the conceptacle is sulfur rich (226 mM) compared with the ECM of the medulla (8 mM). This analysis supports the importance of osmotic modification of the ECM during gamete release and demonstrates that the receptacle is a dynamic signaling organ.     

Adenosine-enhanced ischemic preconditioning: adenosine receptor involvement during ischemia and reperfusion

Adenosine-enhanced ischemic preconditioning (APC) extends the cardioprotection of ischemic preconditioning (IPC) by both significantly decreasing myocardial infarct size and significantly enhancing postischemic functional recovery. In this study, the role of adenosine receptors during ischemia-reperfusion was determined. Rabbit hearts (n = 92) were used for Langendorff perfusion. Control hearts were perfused for 180 min, global ischemia hearts received 30-min ischemia and 120-min reperfusion, and IPC hearts received 5-min ischemia and 5-min reperfusion before ischemia. APC hearts received a bolus injection of adenosine coincident with IPC. Adenosine receptor (A(1), A(2), and A(3)) antagonists were used with APC before ischemia and/or during reperfusion. GR-69019X (A(1)/A(3)) and MRS-1191/MRS-1220 (A(3)) significantly increased infarct size in APC hearts when administered before ischemia and significantly decreased functional recovery when administered during both ischemia and reperfusion (P < 0.05 vs. APC). DPCPX (A(1)) administered either before ischemia and/or during reperfusion had no effect on APC cardioprotection. APC-enhanced infarct size reduction is modulated by adenosine receptors primarily during ischemia, whereas APC-enhanced postischemic functional recovery is modulated by adenosine receptors during both ischemia and reperfusion.     

Apical Vesicles in Growing Bud Cells of Heterobasidiomycetous Yeasts

Clusters of cytoplasmic vesicles resembling those in growing hyphal apices of mycelial fungi are found near the tips of buds in three heterobasidiomycetous yeasts, Rhodotorula glutinis, Candida scottii, and Sporobolomyces salmonicolor.