Molecular Characterization of the α-Subunit of Na+/K+ ATPase from the Euryhaline Barnacle Balanus improvisus Reveals Multiple Genes and Differential Expression of Alternative Splice Variants

The euryhaline bay barnacle Balanus improvisus has one of the broadest salinity tolerances of any barnacle species. It is able to complete its life cycle in salinities close to freshwater (3 PSU) up to fully marine conditions (35 PSU) and is regarded as one of few truly brackish-water species. Na⁺/K⁺ ATPase (NAK) has been shown to be important for osmoregulation when marine organisms are challenged by changing salinities, and we therefore cloned and examined the expression of different NAKs from B. improvisus. We found two main gene variants, NAK1 and NAK2, which were approximately 70% identical at the protein level. The NAK1 mRNA existed in a long and short variant with the encoded proteins differing only by 27 N-terminal amino acids. This N-terminal stretch was coded for by a separate exon, and the two variants of NAK1 mRNAs appeared to be created by alternative splicing. We furthermore showed that the two NAK1 isoforms were differentially expressed in different life stages and in various tissues of adult barnacle, i.e the long isoform was predominant in cyprids and in adult cirri. In barnacle cyprid larvae that were exposed to a combination of different salinities and pCO₂ levels, the expression of the long NAK1 mRNA increased relative to the short in low salinities. We suggest that the alternatively spliced long variant of the Nak1 protein might be of importance for osmoregulation in B. improvisus in low salinity conditions.     

Spitzenk?rper,Exocyst, and Polarisome Components in Candida albicans Hyphae Show Different Patterns of Localization and Have Distinct Dynamic Properties

During the extreme polarized growth of fungal hyphae, secretory vesicles are thought to accumulate in a subapical region called the Spitzenkörper. The human fungal pathogen Candida albicans can grow in a budding yeast or hyphal form. When it grows as hyphae, Mlc1 accumulates in a subapical spot suggestive of a Spitzenkörper-like structure, while the polarisome components Spa2 and Bud6 localize to a surface crescent. Here we show that the vesicle-associated protein Sec4 also localizes to a spot, confirming that secretory vesicles accumulate in the putative C. albicans Spitzenkörper. In contrast, exocyst components localize to a surface crescent. Using a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments and cytochalasin A to disrupt actin cables, we showed that Spitzenkörper-located proteins are highly dynamic. In contrast, exocyst and polarisome components are stably located at the cell surface. It is thought that in Saccharomyces cerevisiae exocyst components are transported to the cell surface on secretory vesicles along actin cables. If each vesicle carried its own complement of exocyst components, then it would be expected that exocyst components would be as dynamic as Sec4 and would have the same pattern of localization. This is not what we observe in C. albicans. We propose a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.Polarized growth of fungi requires that a supply of secretory vesicles is delivered along cytoskeletal tracks to the site of cell expansion (for reviews, see references 13, 29, 30, and 31). Fusion of these membrane-bound vesicles with the plasma membrane allows the necessary expansion of the plasma membrane and releases the enzymes and raw materials for the synthesis of new cell wall material and the remodeling necessary to allow this newly synthesized material to be inserted into the existing cell wall. The process of polarized growth has been extensively studied in the budding yeast Saccharomyces cerevisiae and provides a model for studying the process in other fungi (for a review, see reference 20). Post-Golgi vesicles travel to sites of polarized growth along actin cables (23). Actin cables are nucleated at sites of polarized growth by the formin Bni1 facilitated by a multiprotein complex called the polarisome, which consists of Spa2, Bud6, and Pea1(5, 22, 24, 27). The motive force for vesicle transport is provided by Myo2, a class V myosin, complexed to its regulatory light chain Mlc1 (22, 26). At the plasma membrane, secretory vesicles dock with a second multiprotein complex called the exocyst before fusion with the plasma membrane (14, 15, 32, 33), mediated by v-SNARES on the vesicle and t-SNARES on the membrane. The exocyst is an octomeric complex composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (21). It is thought that Sec3 and a fraction of the Exo70 pool are localized at sites of polarized growth independently of the actin cytoskeleton (3, 6). The other exocyst subunits and the remainder of the Exo70 pool are thought to be transported to sites of polarized growth on secretory vesicles, where together with Sec3 and Exo70 they form the exocyst complex (3). Secretory vesicles exit the Golgi apparatus, travel toward sites of polarized growth, and dock with the exocyst by use of the Rab-type GTPase Sec4 in its GTP-bound form, which is activated by its GEF, Sec2 (12, 19, 35, 36). In the S. cerevisiae cell cycle, polarized growth is initially directed toward the bud tip in young buds (17). Growth subsequently becomes isotropic in larger buds before being directed toward the mother bud neck during cytokinesis at the end of the cell cycle. Accordingly, polarisome and exocyst components localize to the tips of young buds (7, 27, 28).The rate of hyphal tip extension is much greater than that of the growth of a yeast or pseudohyphal bud. In rich yeast extract-peptone-dextrose (YEPD) medium, Candida albicans hyphae extend at the rate of 0.25 μm min⁻¹, compared to 0.0625 μm min⁻¹ in yeast buds and 0.125 μm min⁻¹ in pseudohyphal cells (P. Sudbery unpublished observations). In hyphae of filamentous fungi, a structure called a Spitzenkörper is present at the tip, which is rich in secretory vesicles (8, 9, 11, 29, 34). It is believed that the Spitzenkörper acts as a vesicle supply center (VSC) (1). This model proposes that the Spitzenkörper is maintained at a fixed distance from the hyphal tip. Vesicles radiate out in equal directions to fuse with the plasma membrane, so that more vesicles per unit area fuse with the hyphal tip itself than with other parts of the hyphae. Mathematical modeling shows that this explains the distinctive shape of hyphal tips.In order to investigate the mechanism of polarized growth in the hyphae of Candida albicans, we previously determined the localization of Mlc1-yellow fluorescent protein (YFP) and the polarisome components Bud6-YFP and Spa2-YFP (4). We found that in hyphae, polarisome components localized to a surface crescent, as they did in young yeast buds and the tips of elongated pseudohyphal buds. However, in hyphae Mlc1-YFP localized to a bright spot, which at least in some hyphae was clearly inside the tip, rather than at the surface, and which appeared spherical in three-dimensional reconstructions. We concluded that this represented a Spitzenkörper. In some hyphae Mlc1-YFP also localized to a surface crescent, similar to the pattern displayed by polarisome components. This observation suggested that the Spitzenkörper and polarisome were separate structures, both of which were present at hyphal tips, but that only the polarisome was present at the bud tips of pseudohyphae and yeast. Moreover, the dual localization of Mlc1-YFP to a crescent and a spot suggested that Mlc1 may be present in both structures.While S. cerevisiae has proved to be an excellent model to investigate the molecular genetics of polarized growth, it is less optimal to study the spatial organization of the molecular components because polarized growth of the bud is restricted to a short period after bud emergence when the nascent bud is small. Thus, there has been little effort to investigate the fine detail of the spatial organization of the different components of the polarization machinery beyond noting that they localize to sites of polarized growth. In this study we exploited the opportunities afforded by the continuous polarized growth of C. albicans hyphae to clarify the relationship between the Spitzenkörper, polarisome, and exocyst, which cooperate to mediate the extreme polarized growth of hyphae. We show that the vesicle-associated marker Sec4 also localizes to a Spitzenkörper-like structure, confirming the existence of a vesicle-rich area corresponding to a Spitzenkörper at the hyphal tip. We show that exocyst components such as Sec3, Sec6, Sec8, Exo70, and Exo84 localize to a surface crescent, so the exocyst, like the polarisome, is also a spatially separate structure from the Spitzenkörper. We used three independent strategies to investigate the dynamic properties of these structures. Fluorescence recovery after photobleaching (FRAP) was used to measure the rate at which new proteins arrived at the tip. Fluorescence loss in photobleaching (FLIP) was used to measure the rate at which proteins exited the tip. Cytochalasin A was used to disrupt actin cables, allowing the persistence of proteins at the tip to be measured after the supply of new proteins was blocked. In each case we found that Spitzenkörper components Sec4, Sec2, and Mlc1 were highly dynamic, while the polarisome component Spa2 was stable. Intriguingly, exocyst components showed intermediate dynamic properties, suggesting that they are delivered to the tip on vesicles but that not all vesicles carry a complement of exocyst components. We suggest that these data are consistent with a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.     

Biophysical characterization of α-amylase inhibitor Parvulustat (Z-2685) and comparison with Tendamistat (HOE-467)

Parvulustat is a small, highly active proteinaceous α-amylase inhibitor whose high-resolution NMR structure was recently solved in Frankfurt. Here, we present its biochemical and biophysical characterization. Several spectroscopic methods such as UV, fluorescence and CD were utilized to extract conformational changes upon modification of pH, temperature and chemical denaturant. Parvulustat revealed native like behavior over a wide range of denaturizing agents as reflected in terms of activity and thermodynamic data. In addition, spectroscopic and thermodynamic properties of Parvulustat were compared to the well-characterized Tendamistat. Despite the overall structural similarity, the thermodynamic stability of the two proteins is different. Our analysis led to the conclusion that Parvulustat is even more stable than Tendamistat. Furthermore, investigations on three C-terminally truncated Parvulustat derivatives indicate that the higher stability is caused by the long flexible C-terminus.     

Kinetics of Inhibition of Green Crab (Scylla Serrata) Alkaline Phosphatase by Sodium (2,2′-bipyridine) Oxodiperoxovanadate

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The kinetics of inhibition of the enzyme by sodium (2, 2′-bipyridine) oxodiperoxovanadate, pV(bipy), has been studied. The time course of the hydrolysis of p-nitrophenyl-phosphate catalyzed by the enzyme in the presence of different pV(bipy) concentrations showed that at each pV(bipy) concentration, the rate decreased with increasing time until a straight line was approached, the straight line slopes are the same for all concentrations. The results suggest that the inhibition of the enzyme by pV(bipy) is a slow, reversible reaction with fractional remaining activity. The microscopic rate constants are determined for the reaction of inhibitor with the enzyme.     

Properties of 5′-nucleotidase from nodules of pigeonpea (Cajanus cajan)

5′-Nucleotidase from pigeonpea nodules has been resolved into two forms, N-I and N-II, having M,s of 52 000 and 119 000, respectively. Both forms had pH optima in the acidic range (between pH 5.2 and 5.7) with either CMP, GMP, XMP, IMP or AMP as the substrate. Up to pH 6.6, both forms showed higher activity with CMP followed by GMP, XMP, IMP and AMP, respectively. However, the activity changed with pH in the alkaline range making the enzyme relatively more active with purine nucleotides. Neither of the forms had a requirement for any of the metal ions tested. Fe³⁺ inhibited the enzyme activity; the inhibition at 5, 10 and 15 mM concentrations being 11, 43 and 47%, respectively with N-I and 14,47 and 52%, respectively with N-II. K_m values for AMP, IMP, GMP, CMP and XMP were 0.10, 0.18, 0.40, 0.40 and 0.77 mM, respectively with N-I and 0.12, 0.20, 0.40, 0.40 and 0.99 mM, respectively with N-II. The enzyme was inhibited non-competitively by adenosine and inosine; K_i values being 1.78, 0.25 and 0.30; 3.50, 2.12 and 0.75 mM, respectively with AMP, IMP and XMP as the substrate.     

Effects of (±)-kavain on inactivation of voltage-operated Na+ channels

Effects of a kava-pyrone (±)-kavain on fast inactivation of Na⁺ channels were studied in experiments on isolated neurons from the rat hippocampus. (±)-Kavain was found to block Na⁺ channels, and its effect was voltage-dependent. At the holding potentials of –100 and –80 mV, IC₅₀ for (±)-kavain was 744.9 and 178.8 µM, respectively. The inactivation characteristic of Na⁺ channels was satisfactorily described with the Boltzmann's equation both in the control and under (±)-kavain application. (±)-Kavain at a 330 µM concentration shifted theV _1/2 toward more negative values by 14.4 mV and concurrently modified the slope factor: the latter was 5.7 mV in the control, while under the influence of 330 µM (±)-kavain it reached 6.7 mV. In agreement with Hille's hypothesis of a modulated receptor, inactivated Na⁺ channels demonstrated an increased sensitivity to kavain. (±)-Kavain effects resulted in an increase in the rate of depolarization-related fast inactivation, while the process of recovery from inactivation became slower when the membrane was hyperpolarized. Our data show that under the (±)-kavain effect the probability of the inactivated state of Na⁺ channels increases, and the state of fast inactivation is stabilized.Neirofiziologiya/Neurophysiology, Vol. 28, No. 4/5, pp. 218–224, July–October, 1996.     

Have we overestimated the benefit of human(ized) antibodies?

The infusion of animal-derived antibodies has been known for some time to trigger the generation of antibodies directed at the foreign protein as well as adverse events including cytokine release syndrome. These immunological phenomena drove the development of humanized and fully human monoclonal antibodies. The ability to generate human(ized) antibodies has been both a blessing and a curse. While incremental gains in the clinical efficacy and safety for some agents have been realized, a positive effect has not been observed for all human(ized) antibodies. Many human(ized) antibodies trigger the development of anti-drug antibody responses and infusion reactions. The current belief that antibodies need to be human(ized) to have enhanced therapeutic utility may slow the development of novel animal-derived monoclonal antibody therapeutics for use in clinical indications. In the case of murine antibodies, greater than 20% induce tolerable/negligible immunogenicity, suggesting that in these cases humanization may not offer significant gains in therapeutic utility. Furthermore, humanization of some murine antibodies may reduce their clinical effectiveness. The available data suggest that the utility of human(ized) antibodies needs to be evaluated on a case-by-case basis, taking a cost-benefit approach, taking both biochemical characteristics and the targeted therapeutic indication into account.Key words: immunogenicity, human anti-mouse antibody, cytokine release syndrome     

Have we overestimated the benefit of human(ized) antibodies?

The infusion of animal-derived antibodies has been known for some time to trigger the generation of antibodies directed at the foreign protein as well as adverse events including cytokine release syndrome. These immunological phenomena drove the development of humanized and fully human monoclonal antibodies. The ability to generate human(ized) antibodies has been both a blessing and a curse. While incremental gains in the clinical efficacy and safety for some agents have been realized, a positive effect has not been observed for all human(ized) antibodies. Many human(ized) antibodies trigger the development of anti-drug antibody responses and infusion reactions. The current belief that antibodies need to be human(ized) to have enhanced therapeutic utility may slow the development of novel animal-derived monoclonal antibody therapeutics for use in clinical indications. In the case of murine antibodies, greater than 20% induce tolerable/negligible immunogenicity, suggesting that in these cases humanization may not offer significant gains in therapeutic utility. Furthermore, humanization of some murine antibodies may reduce their clinical effectiveness. The available data suggest that the utility of human(ized) antibodies needs to be evaluated on a case-by-case basis, taking a cost-benefit approach, taking both biochemical characteristics and the targeted therapeutic indication into account.     

Chronic Exposure of NG108-15 Cells to Amyloid β Peptide (Aβ1–42) Abolishes Calcium Influx via N-Type Calcium Channels

We investigated whether amyloid--peptide (A_1–42) has an effect on the elevations of the intracellular concentration of Ca²⁺ ions ([Ca²⁺]_i) induced by depolarizations of NG108-15 cells and on related Ca²⁺ channels. A_1–42 (10-1000 nM) had no immediate effect on depolarization-induced [Ca²⁺]_i elevations. [Ca²⁺]_i increases were slightly diminished in cells grown in the presence of 100 or 1000 nM A_1–42. Nifedipine (1 M) reduced these elevations equally in cells grown in the absence or presence of A_1–42. In contrast, the ability of -conotoxin GVIA to diminish the depolarization-induced [Ca²⁺]_i responses became lost in cells grown in the presence of 100 nM A_1–42. This indicates that the influx of calcium through the N-type Ca²⁺ channels was compromised by the chronic exposure of cells to a submicromolar concentration of A_1–42, presumably because of impairement of their function or diminished expression. This may be important in the pathogeny of Alzheimer's dementia in view of the pivotal role of N-type Ca²⁺ channels in neurotransmitter release.     

Tabula rasa in the Patagonian Channels? The phylogeography of Oreobolus obtusangulus (Cyperaceae)

The extent of the Pleistocene glaciations in the Patagonian Channel region (southwesternmost South America) and their impact on the vegetation there are largely unknown. Whether the regional flora was wiped out completely (tabula rasa) or survived in ice‐free pockets (in situ survival) is still an open question. The molecular imprint of either scenario should still be visible in extant populations. Therefore, DNA sequence data of Oreobolus obtusangulus Gaudich. (Cyperaceae) were analysed. This species is an abundant constituent of Patagonian cushion peat bogs, one of the Patagonian Channel region's major vegetation types. Three hundred and eighty‐four individuals from 48 populations were sequenced for two chloroplast (ycf3‐psaA and trnQ^UUG‐psbK intergenic spacers) and 14 nuclear loci containing simple sequence repeats (SSRs; microsatellites). Phylogenetic reconstructions and the geographic distribution of genetic diversity revealed that the species was split into three main lineages whose general distributions comprise three separate major regions, that is, south‐central Chile, Fuego‐Patagonia and the East Patagonian Andes, which probably constitute glacial refugia. Postglacial migration fronts formed a suture zone with high levels of genetic diversity in the Northwest Patagonian Andes, where remnants of a supposedly ancestral lineage were also found to be locally restricted to a single population (Huinay). The heavily glaciated Patagonian Channels were likely recolonized from the northwest, and partly from the south. Although the westernmost Patagonian Channel population (Estero Bachem) harboured private SSR alleles (singletons) and showed slightly elevated genetic diversity, it remained unclear whether this population actually survived in situ. This study helps fill a major gap in reconstructing the Pleistocene vegetation history of West and Andean Patagonia.     

Small Liposomes Accelerate the Fibrillation of Amyloid β (1–40)

The deposition of amyloid β (Aβ) peptides is a pathological hallmark of Alzheimer disease. Aβ peptides were previously considered to interact specifically with ganglioside-containing membranes. Several studies have suggested that Aβ peptides also bind to phosphatidylcholine membranes, which lead to deformation of membranes and fibrillation of Aβ. Moreover, the role of membrane curvature, one type of deformation produced by binding of proteins to a membrane, in the binding and fibrillation of Aβ remains unclear. To clearly understand the relationship between the binding, consequent membrane deformation, and fibrillation of Aβ, we examined the amyloid fibrillation of Aβ-(1–40) in the presence of liposomes of various sizes. Membrane curvature increased with a decrease in the size of the liposomes. We used liposomes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine to eliminate electrostatic effects. The results obtained showed that liposomes of smaller sizes (≤50 nm) significantly accelerated the nucleation step, thereby shortening the lag time of fibrillation. On the other hand, liposomes of larger sizes decreased the amount of fibrils but did not notably affect the lag time. The morphologies of fibrils, which were monitored by total internal reflection fluorescence microscopy, atomic force microscopy, and transmission electron microscopy, revealed that the length of Aβ-(1–40) fibrils became shorter and the amount of amorphous aggregates became larger as liposomes increased in size. These results suggest that the curvature of membranes coupled with an increase in water-accessible hydrophobic regions is important for binding and concentrating Aβ monomers, leading to amyloid nucleation. Furthermore, amyloid fibrillation on membranes may compete with non-productive binding to produce amorphous aggregates.     

Single-channel monitoring of reversible L-type Ca(2+) channel Ca(V)α(1)-Ca(V)β subunit interaction

Voltage-dependent Ca(2+) channels are heteromultimers of Ca(V)α(1) (pore), Ca(V)β- and Ca(V)α(2)δ-subunits. The stoichiometry of this complex, and whether it is dynamically regulated in intact cells, remains controversial. Fortunately, Ca(V)β-isoforms affect gating differentially, and we chose two extremes (Ca(V)β(1a) and Ca(V)β(2b)) regarding single-channel open probability to address this question. HEK293α(1C) cells expressing the Ca(V)1.2 subunit were transiently transfected with Ca(V)α(2)δ1 alone or with Ca(V)β(1a), Ca(V)β(2b), or (2:1 or 1:1 plasmid ratio) combinations. Both Ca(V)β-subunits increased whole-cell current and shifted the voltage dependence of activation and inactivation to hyperpolarization. Time-dependent inactivation was accelerated by Ca(V)β(1a)-subunits but not by Ca(V)β(2b)-subunits. Mixtures induced intermediate phenotypes. Single channels sometimes switched between periods of low and high open probability. To validate such slow gating behavior, data were segmented in clusters of statistically similar open probability. With Ca(V)β(1a)-subunits alone, channels mostly stayed in clusters (or regimes of alike clusters) of low open probability. Increasing Ca(V)β(2b)-subunits (co-)expressed (1:2, 1:1 ratio or alone) progressively enhanced the frequency and total duration of high open probability clusters and regimes. Our analysis was validated by the inactivation behavior of segmented ensemble averages. Hence, a phenotype consistent with mutually exclusive and dynamically competing binding of different Ca(V)β-subunits is demonstrated in intact cells.     

Isolation and Properties of an α-Amylase Inhibitor (0.53) from Wheat (Triticum aestivum)

A droplet gel-entrapping method used for enzyme immobilization was improved to simplify the procedure and to increase the enzyme stability. This immobilization technique is suitable for coupled enzyme reactions requiring cofactors. Leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) were freeze-dried with bovine serum albumin, dextrin and stabilizers. The freeze-dried enzyme powder was suspended in a methylcellosolve solution containing polyethyleneglycol(#4000)diacrylate, N,N′-methylenebisacrylamide and 2-hydroxyethylacrylate, and the suspension was gelled with initiators. The gel was cut up and the pieces were washed in a buffer to remove the methylcellosolve and the dextrin inside. The maximum conversion ratio for a LeuDH-FDH gel column was determined to be 99.8% by means of the recycling reaction. On longterm operation at 30 °C for leucine production, the initial conversion ratio (7.2%) gradually decreased to 6.6% over the first 10 days. However, the conversion ratio remained almost constant after the 10th day. The effects of flow rate, temperature, pH, and the concentrations of formate, α- ketoisocaproate, ammonium and NAD on the leucine productivity with the gel column were also investigated.     

Biophysical and Structural Characterization of Novel RAS-Binding Domains (RBDs) of PI3Kα and PI3Kγ

Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate to generate a key lipid second messenger, phosphatidylinositol 3,4,5-bisphosphate. PI3Kα and PI3Kγ require activation by RAS proteins to stimulate signaling pathways that control cellular growth, differentiation, motility and survival. Intriguingly, RAS binding to PI3K isoforms likely differ, as RAS mutations have been identified that discriminate between PI3Kα and PI3Kγ, consistent with low sequence homology (23%) between their RAS binding domains (RBDs). As disruption of the RAS/PI3Kα interaction reduces tumor growth in mice with RAS- and epidermal growth factor receptor driven skin and lung cancers, compounds that interfere with this key interaction may prove useful as anti-cancer agents. However, a structure of PI3Kα bound to RAS is lacking, limiting drug discovery efforts. Expression of full-length PI3K isoforms in insect cells has resulted in low yield and variable activity, limiting biophysical and structural studies of RAS/PI3K interactions. This led us to generate the first RBDs from PI3Kα and PI3Kγ that can be expressed at high yield in bacteria and bind to RAS with similar affinity to full-length PI3K. We also solved a 2.31 Å X-ray crystal structure of the PI3Kα-RBD, which aligns well to full-length PI3Kα. Structural differences between the PI3Kα and PI3Kγ RBDs are consistent with differences in thermal stability and may underly differential RAS recognition and RAS-mediated PI3K activation. These high expression, functional PI3K RBDs will aid in interrogating RAS interactions and could aid in identifying inhibitors of this key interaction.     

Intracellular and cell wall associated (1 → 3) β glucanases of Saprolegnia

Summary Fractionation of proteins of mycelial cell free extracts from Saprolegnia monoica revealed the presence of two different (1.3) glucanases. The most important fraction exhibited activity against laminarin and p. nitrophenyl BD. glucopyranoside. The other was active on both laminarin and oxidized laminarin. This endo-glucanase represented the main part of glucanase activities released during cell wall autolysis. Properties and cellular distribution of these enzymes are discussed in respect to their morphogenetic role in hyphal differentiation.     

Effects of Hydrophobic Macromolecular Crowders on Amyloid β (16–22) Aggregation

In Alzheimer’s disease (AD), the amyloid β (Aβ) peptide aggregates in the brain to form progressively larger oligomers, fibrils, and plaques. The aggregation process is strongly influenced by the presence of other macromolecular species, called crowders, that can exert forces on the proteins. One very common attribute of macromolecular crowders is their hydrophobicity. We examined the effect of hydrophobic crowders on protein aggregation by using discontinuous molecular dynamics (DMD) simulations in combination with an intermediate resolution protein model, PRIME20. The systems considered contained 48 Aβ (16–22) peptides and crowders with diameters of 5 Å, 20 Å, and 40 Å, represented by hard spheres or spheres with square-well/square-shoulder interactions, at a crowder volume fraction of ϕ = 0.10. Results show that low levels of crowder hydrophobicity are capable of increasing the fibrillation lag time and high levels of crowder hydrophobicity can fully prevent the formation of fibrils. The types of structures that remain during the final stages of the simulations are summarized in a global phase diagram that shows fibril, disordered oligomer, or β-sheet phases in the space spanned by crowder size and crowder hydrophobicity. In particular, at high levels of hydrophobicity, simulations with 5 Å crowders result in only disordered oligomers and simulations with 40 Å crowders result in only β-sheets. The presence of hydrophobic crowders reduces the antiparallel β-sheet content of fibrils, whereas hard sphere crowders increase it. Finally, strong hydrophobic crowders alter the secondary structure of the Aβ (16–22) monomers, bending them into a shape that is incapable of forming ordered β-sheets or fibrils. These results qualitatively agree with previous theoretical and experimental work.