Random Motion of Chromatin Is Influenced by Lamin A Interconnections

Using fluorescence correlation spectroscopy in single-plane illumination microscopy, we investigated the dynamics of chromatin in interphase mouse adult fibroblast cell nuclei under the influence of the intermediate filament protein lamin A. We find that 1) lamin A-eGFP and histone H2A-mRFP show significant comobility, indicating that their motions are clearly interconnected in the nucleus, and 2) that the random motion of histones H2A within the chromatin network is subdiffusive, i.e., the effective diffusion coefficient decreases for slow timescales. Knocking out lamin A changes the diffusion back to normal. Thus, lamin A influences the dynamics of the entire chromatin network. Our conclusion is that lamin A plays a central role in determining the viscoelasticity of the chromatin network and helping to maintain local ordering of interphase chromosomes.     

Ubiquitin-Modulated Phase Separation of Shuttle Proteins: Does Condensate Formation Promote Protein Degradation?

Liquid-liquid phase separation (LLPS) has recently emerged as a possible mechanism that enables ubiquitin-binding shuttle proteins to facilitate the degradation of ubiquitinated substrates via distinct protein quality control (PQC) pathways. Shuttle protein LLPS is modulated by multivalent interactions among their various domains as well as heterotypic interactions with polyubiquitin chains. Here, the properties of three different shuttle proteins (hHR23B, p62, and UBQLN2) are closely examined, unifying principles for the molecular determinants of their LLPS are identified, and how LLPS is connected to their functions is discussed. Evidence supporting LLPS of other shuttle proteins is also found. In this review, it is proposed that shuttle protein LLPS leads to spatiotemporal regulation of PQC activities by mediating the recruitment of PQC machinery (including proteasomes or autophagic components) to biomolecular condensates, assembly/disassembly of condensates, selective enrichment of client proteins, and extraction of ubiquitinated proteins from condensates in cells.     

Kinetics of Phase Separation of Oat β-Glucan/Whey Protein Isolate Binary Mixtures

The kinetics of phase separation and microstructure of oat β-glucan/whey protein binary mixtures varying in concentration (4–16% w/v protein, 0.3–1.2% w/v β-glucan) and β-glucan molecular weight (1.3 × 10⁶, 640 × 10³, 180 × 10³, and 120 × 10³ g/mol) was investigated by turbidimetry and fluorescent microscopy. The phase separation of the mixed systems was followed at pH 7.0 and at room temperature under quiescent conditions. Application of first principles revealed that phase separation of the systems follows first-order kinetics. Acceleration of the phase-separation process was observed with increase of β-glucan concentration for the three lowest-MW samples but the highest molecular weight (1.3 × 10⁶ g/mol) exhibited the opposite trend. Changes in the polysaccharide molecular weight resulted in considerable differences in β-glucan aggregate morphology in the mixed systems. The change in the continuity of the mixed system from polysaccharide-, to bi-, to protein-continuous was confirmed for a wide range of mixed systems differing in biopolymer concentration, and β-glucan molecular weight.     

Formation of 20β-dihydrosteroids by anaerobic bacteria

Cortisol was metabolized to a variety of products, among them small amounts of cortol by fecal flora of humans and rats.A microorganism. Bifidobacterium adolescentis, isolated from both sources, synthesized a 20-hydroxysteroid dehydrogenase which reduced cortisol to 20β-dihydrocortisol. The metabolite was reduced to cortol by Clostridium paraputrificum. The 20-hydroxysteroid dehydrogenase showed a wide substrate specificity; it was independent of the 4-ene and the configuration at C-3, C-11, C-17 and C-21. Cortol was resistant to any further alteration by human fecal flora, i.e. it is a metabolic end product. As expected. B. adolescenris effectively prevented 21-dehydroxylation of cortisol by Eubacterium lentum.     

Formation of γ-glutamylglycyglycine by extracellular glutaminase of Aspergillus oryzae

γ-Glutamylglycylglycine (γ-GluGlyGly) was formed through the γ-glutamyltranspeptidase (GGT) reaction catalyzed by glutaminase in a water extract of wheat bran koji obtained with Aspergillus oryzae MA-27-IM. The yield of γ-GluGlyGly was about 18% from l-glutamine in a reaction mixture containing 50 mM l-glutamine, 50 mM glycylglycine, and the extract (0.1 unit ml as GGT activity) in a 100 mM Tris-HCl buffer solution (pH 7.2), which was incubated for 7 h at 30°C. The γ-GluGlyGly formed was purified by ion exchange chromatographies, and the identified by chemical and enzymatic methods as well as by infrared and PMR spectroscopic analyses.     

Formation of Three New Trisaccharide-azoxyglycosides by Transglucosylation by Cycad β-Glucosidase

transglucosylation by a β-d-glucosidase from cycad seeds. These azoxyglycosides, named neocycasin H, I, and J, were identified as O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(l→3)-O-β-d-glucopyranoside of methylazoxymethanol (MAM), O-β-d-glucopyranosyl-(1→3)-[O-β-d-glucopyranosyl-(1→6)]-O-β-d-glucopyranoside of MAM, and O-β-d-glucopyranosyl-(1→3)-[O-β-d-xylopyranosyl-(1→6)]-O-β-d-glucopyranoside of MAM, respectively. On the basis of their structures, the mechanism of the formation of these neocycasins is also discussed.     

Separation of trypsin from trypsin α-chymotrypsin mixture by affinity-ultrafiltration

Summary Water-sobuble trypsin specific macroligands were prepared to separate the trypsin -chymotrypsin mixture with affinity-ultrafiltration technique. Soya bean trypsin inhibitor (STI) attached to cyanogen bromideactivated Dextran showed a good selectivity and low non-specific adsorption properties. The experiments performed with STI-Dextran polymer gave a 81% purified trypsin from 50%-50% mixture.     

Amyloid β Oligomeric Species Present in the Lag Phase of Amyloid Formation

Alzheimer’s disease (AD)-associated amyloid β peptide (Aβ) is one of the main actors in AD pathogenesis. Aβ is characterized by its high tendency to self-associate, leading to the generation of oligomers and amyloid fibrils. The elucidation of pathways and intermediates is crucial for the understanding of protein assembly mechanisms in general and in conjunction with neurodegenerative diseases, e.g., for the identification of new therapeutic targets. Our study focused on Aβ42 and its oligomeric assemblies in the lag phase of amyloid formation, as studied by sedimentation velocity (SV) centrifugation. The assembly state of Aβ during the lag phase, the time required by an Aβ solution to reach the exponential growth phase of aggregation, was characterized by a dominant monomer fraction below 1 S and a population of oligomeric species between 4 and 16 S. From the oligomer population, two major species close to a 12-mer and an 18-mer with a globular shape were identified. The recurrence of these two species at different initial concentrations and experimental conditions as the smallest assemblies present in solution supports the existence of distinct, energetically favored assemblies in solution. The sizes of the two species suggest an Aβ42 aggregation pathway that is based on a basic hexameric building block. The study demonstrates the potential of SV analysis for the evaluation of protein aggregation pathways.     

Turing Pattern Formation with Two Kinds of Cells and a Diffusive Chemical

We study a three-variable Turing system with two kinds of cells and a diffusive chemical, considering the formation and maintenance of fish skin patterns with multiple pigment cells. The two types of cells are produced from undifferentiated cells. They inhibit the supply rate of the other cell type, forming local clusters of the same cell type. In addition, the cells of one type can be maintained only in the presence of a diffusive chemical produced by the other cell type, resulting in the coexistence of two cell types in heterogeneous spatial patterns. We assume linear interaction among cells and the chemical, and cell supply rates constrained to be positive or zero. We derive the condition for diffusion-driven instability. In one-dimensional model, we examine how the wavelength of the periodic pattern depends on parameters. In the two-dimensional model, we study the condition for spot, stripe or reversed spot pattern to emerge (pattern selection). We discuss heuristic criteria for the pattern selection.     

Enzyme-Induced Formation of β-Lactoglobulin Fibrils by AspN Endoproteinase

This paper describes a low temperature, enzymatic route to induce fibrillar structures in a protein solution. The route comprises two steps. First, β-lactoglobulin was hydrolyzed into peptides at pH 8 and 37 °C with the enzyme AspN endoproteinase, which resulted in the formation of random aggregates. After hydrolysis, the pH was lowered to 2. As a result, long fibrillar aggregates were formed which was observed using transmission electron microscopy and Thioflavin T fluorescence measurements.     

Controlling Liquid–Liquid Phase Separation of Cold-Adapted Crystallin Proteins from the Antarctic Toothfish

Liquid–liquid phase separation (LLPS) of proteins is important to a variety of biological processes both functional and deleterious, including the formation of membraneless organelles, molecular condensations that sequester or release molecules in response to stimuli, and the early stages of disease-related protein aggregation. In the protein-rich, crowded environment of the eye lens, LLPS manifests as cold cataract. We characterize the LLPS behavior of six structural γ-crystallins from the eye lens of the Antarctic toothfish Dissostichus mawsoni, whose intact lenses resist cold cataract in subzero waters. Phase separation of these proteins is not strongly correlated with thermal stability, aggregation propensity, or cross-species chaperone protection from heat denaturation. Instead, LLPS is driven by protein–protein interactions involving charged residues. The critical temperature of the phase transition can be tuned over a wide temperature range by selective substitution of surface residues, suggesting general principles for controlling this phenomenon, even in compactly folded proteins.     

Formation of α- and β-conidia by Phaeocytostroma ambiguum

The formation of conidia in Phaeocytostroma ambiguum on different media and conditions was investigated in this study. Carnation leaf agar (CLA) and a 12 h photoperiod (24/18 °C) provided excellent conditions for the promotion of rapid formation of both alpha (α) and beta (β) conidia in a number of P. ambiguum isolates. The dimensions of α- and β-conidia amounted to 6.0–19.6 × 3.8–7.5 μm and 6.0–24.9 × 1.1–2.6 μm, respectively. They were produced on short or elongate, simple and branched conidiophores. β-conidia have not been described before in P. ambiguum. Intermediate conidia were rarely found. This revised version was published online in August 2006 with corrections to the Cover Date.     

Expression and Secretion of Bifidobacterium adolescentis Amylase by Bifidobacterium Longum

Bifidobacterium adolescentis Int-57 (INT57), isolated from human feces, secretes an amylase. We have shot-gun cloned, sequence analyzed and expressed the gene encoding this amylase in B. longum. The sequenced 2477 bp fragment was homologous to other extracellular amylases. The encoded protein was predicted to be composed of 595 amino acids with a molecular weight of 64 kDa, and was designated AmyB. Highly conserved amylase domains were found in AmyB. The signal sequence and cleavage site was predicted by sequence analysis. AmyB was subcloned into pBES2, a novel E. coli–Bifidobacterium shuttle vector, to construct pYBamy59. Subsequently, B. longum, with no apparent amylase activity, was transformed with pYBamy59. More than 90% of the amylase activity was detected in the culture broth. This approach may open the way for the development of more efficient expression and secretion systems for Bifidobacterium. Both authors contributed equally Received 17 June 2005; Revisions requested 13 July 2005 and 26 September 2005; Revisions received 12 September 2005 and 8 November 2005; Accepted 11 November 2005     

Separation of recombinant β-glucuronidase from transgenic tobacco by aqueous two-phase extraction

Transgenic plants hold many promises as viable production hosts for therapeutic recombinant proteins. Many efforts have been devoted to increase the expression level of the proteins, but the efforts for developing economic processes to purify those proteins are lacking. In this report, aqueous two-phase extraction (ATPE) was investigated as an alternative for the separation of an acidic recombinant protein, β-glucuronidase (rGUS), from transgenic tobacco. Screening experiments by fractional factorial designs showed that PEG concentration and ionic strength of the system significantly affected the partitioning of native tobacco proteins and GUS. Response surface methodology was used to determine an optimized aqueous two-phase system for the purification of rGUS from transgenic tobacco. In a 13.4% (w/w) PEG 3400/18% (w/w) potassium phosphate system, 74% of the rGUS was recovered in the top PEG-rich phase while more than 90% of the native tobacco proteins were removed in the interphase and the bottom phase. A purification factor of about 20 was achieved in this process. The most important impurity from tobacco, Rubisco, was largely removed from the rGUS in the recovered phase.