Protein Insolubilization Research Articles

Lens Cytoskeleton: An Update on the Etiopathogenesis of Human Cataracts.

A cataract is a loss of the transparency of a normal crystalline lens. Multiple factors, including age as the major risk factor for cataracts, can disturb the transparency of the crystalline lens due to cumulative damage from environmental insults to proteins, particularly crystallins. Lens proteins do not turnover, and crystallins undergo extensive post-translational modifications (PTMs) with age in order to interact with each other and maintain their soluble basis for lens transparency. These PTMs include truncation, oxidation, deamidation, acetylation, phosphorylation, and glycosylation. Cataract formation, apart from protein PTMs, involves protein crosslinking, protein insolubilization, and aggregation. Oxidation is a key feature in age-related cataract formation. Due to the role of genetic and environmental factors, as well as its variable clinical presentation, we consider cataracts to be a multifactorial disease. The preliminary results of our study indicate that proteins implicated in the pathway of a structural constituent of the eye lens (BFSP1, BFSP2, CRYAA, CRYAB, CRYBA, CRYBB, CRYGC, CRYGD, CRYGS, KRTs, and VIM), together with AQP1 and AQP5, may also be involved in lens aging.

Transport proteins and their differential roles in the accumulation of phenanthrene in wheat

The study focuses on the uptake, accumulation, and translocation of polycyclic aromatic hydrocarbons (PAHs) in cereals, specifically exploring the role of peroxidase (UniProt accession: A0A3B5XXD0, abbreviation: PX1) and unidentified protein (UniProt accession: A0A3B6LUC6, abbreviation: UP1) in phenanthrene solubilization within wheat xylem sap. This research aims to clarify the interactions between these proteins and phenanthrene. Employing both in vitro and in vivo analyses, we evaluated the solubilization capabilities of recombinant transport proteins for phenanthrene and examined the relationship between protein expression and phenanthrene concentration. UP1 displayed greater transport efficiency, while PX1 excelled at lower concentrations. Elevated PX1 levels contributed to phenanthrene degradation, marginally diminishing its transport. Spectral analyses and molecular dynamics simulations validated the formation of stable protein-phenanthrene complexes. The study offers crucial insights into PAH-related health risks in crops by elucidating the mechanisms of PAH accumulation facilitated by transport proteins.

Characterization of protein aggregates in cream and skimmed human milk after heat and high-pressure pasteurization treatments.

Preservation processes applied to ensure microbial safety of human milk (HM) can modify the native structure of proteins and their bioactivities. Consequently, this study evaluated the effect of pasteurization methods (Holder pasteurization, high-temperature short-time (HTST), and high hydrostatic pressure (HHP)) of whole human milk (HM) on protein aggregates in skim milk and cream fractions. For heat-treated whole milk, insoluble protein aggregates at milk fat globule membrane (MFGM) were formed by disulfide and non-covalent bonds, but insoluble skim milk protein aggregates were only stabilized by non-covalent interactions. Contrary to heat treatment, the insolubilization of main proteins at the MFGM of HHP-treated HM was only through non-covalent interactions rather than disulfide bonds. Moreover, only heat treatment induced the insoluble aggregation of ⍺-lactalbumin. Overall, compared to heat treatment, HHP produced a milder effect on protein aggregation, validating the use of this process to better preserve the native state of HM bioactive proteins.

Relationship between intracellular protein denaturation and irreversible inactivation of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles.

To clarify the relationship between irreversible inactivation and intracellular protein denaturation of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles (CO2 MB) treatment, a storage test of S. pastorianus cells treated with CO2 MB was performed, and the effect on the intracellular protein was investigated. In the storage test, the S. pastorianus population, which decreased below the detection limit by CO2 MB treatment at a temperature of 45 and 50°C (MB45 and MB50), and thermal treatment at a temperature of 80°C (T80), remained undetectable during storage for 3 weeks at 25°C. However, 4.1 and 1.3-logs of the S. pastorianus populations, which survived after CO2 MB treatment at temperatures of 35 and 40°C (MB35 and MB40), increased gradually during storage for 3 weeks at 25°C. Insolubilization of intracellular proteins in S. pastorianus increased with increasing the temperature of CO2 MB treatment. Activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) identified as one of the insolubilized proteins increased at MB35 and MB40 than non-treatment but disappeared at MB45 and MB50, and T80. Therefore, it was revealed that S. pastorianus cells inactivated below the detection level by CO2 MB treatment did not regrow and that the denaturation of intracellular proteins of S. pastorianus was caused by CO2 MB and thermal treatments. Furthermore, it was suggested that denaturation of intracellular vital enzymes was an important factor for achieving irreversible inactivation of S. pastorianus by CO2 MB and thermal treatments.

Involvement of increased endoplasmic reticulum stress in the development of cataracts in BALB.NCT-Cpoxnct mice

The BALB.NCT-Cpoxnct is a mutant mouse model for hereditary cataracts. We previously uncovered that the primary cause of the cataracts of BALB.NCT-Cpoxnct is a mutation in the coproporphyrinogen oxidase (Cpox) gene. Because of the mutation, excessive coproporphyrin is accumulated in the BALB.NCT-Cpoxnct lens. In this study, we analyzed the changes in transcriptome and proteins in the lenses of 4- and 12-week-old BALB.NCT-Cpoxnct to further elucidate the molecular etiology of cataracts in this mouse strain. Transcriptome analysis revealed that endoplasmic reticulum (ER) stress was increased in the BALB.NCT-Cpoxnct lens that induced persistent activation of the PERK signaling pathway of the ER stress response. Also, levels of crystallin transcripts and proteins were reduced in the BALB.NCT-Cpoxnct lens. Analysis of proteins disclosed aggregation of crystallins and keratins prior to the manifestation of cataracts in 4-week-old BALB.NCT-Cpoxnct mice. At 12 weeks of age, insoluble crystallins were accumulated in the cataractous BALB.NCT-Cpoxnct lens. Overall, our data suggest the following sequence of events in the BALB.NCT-Cpoxnct lens: accumulated coproporphyrin induces the aggregation of proteins including crystallins. Aggregated proteins increase ER stress that, in turn, leads to the repression of global translation of proteins including crystallins. The decline in the molecular chaperone crystallin aggravates aggregation and insolubilization of proteins. This vicious cycle would eventually lead to cataracts in BALB.NCT-Cpoxnct.

Increased Association of Deamidated \u03b1A-N101D with Lens membrane of transgenic \u03b1AN101D vs. wild type \u03b1A mice: potential effects on intracellular ionic imbalance and membrane disorganization

We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization.MethodsLenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.ResultsCompared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice.ConclusionsThe results show that an increased lens membrane association of αAN101D-−relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

Human cataractous lenses contain cross-links produced by crystallin-derived tryptophanyl and tyrosyl radicals

Protein insolubilization, cross-linking and aggregation are considered critical to the development of lens opacity in cataract. However, the information about the presence of cross-links other than disulfides in cataractous lenses is limited. A potential role for cross-links produced from tryptophanyl radicals in cataract development is suggested by the abundance of the UV light-sensitive Trp residues in crystallin proteins. Here we developed a LC-MS/MS approach to examine the presence of Trp−Trp, Trp−Tyr and Tyr−Tyr cross-links and of peptides containing Trp−2H (−2.0156 Da) in the lens of three patients diagnosed with advanced nuclear cataract. In the proteins of two of the lenses, we characterized intermolecular cross-links between βB2-Tyr153−Tyr104-βA3 and βB2-Trp150−Tyr139-βS. An additional intermolecular cross-link (βB2-Tyr61−Trp200-βB3) was present in the lens of the oldest patient. In the proteins of all three lenses, we characterized two intramolecular Trp−Trp cross-links (Trp123−Trp126 in βB1 and Trp81−Trp84 in βB2) and six peptides containing Trp −2H residues, which indicate the presence of additional Trp−Trp cross-links. Relevantly, we showed that similar cross-links and peptides with modified Trp−2H residues are produced in a time-dependent manner in bovine β-crystallin irradiated with a solar simulator. Therefore, different crystallin proteins cross-linked by crystalline-derived tryptophanyl and tyrosyl radicals are present in advanced nuclear cataract lenses and similar protein modifications can be promoted by solar irradiation even in the absence of photosensitizers. Overall, the results indicate that a role for Trp−Tyr and Trp−Trp cross-links in the development of human cataract is possible and deserves further investigation.

Computational studies on nonenzymatic succinimide-formation mechanisms of the aspartic acid residues catalyzed by two water molecules

In the biological proteins, aspartic acid (Asp) residues are prone to nonenzymatic isomerization via a succinimide (Suc) intermediate. Asp-residue isomerization causes the aggregation and the insolubilization of proteins, and is considered to be involved in various age-related diseases. Although Suc intermediate was considered to be formed by nucleophilic attack of the main-chain amide nitrogen of N-terminal side adjacent residue to the side-chain carboxyl carbon of Asp residue, previous studies have shown that the nucleophilic attack is more likely to proceed via iminol tautomer when the water molecules act as catalysts. However, the full pathway to Suc-intermediate formation has not been investigated, and the experimental analyses for the Asp-residue isomerization mechanism at atomic and molecular levels, such as the analysis of the transition state geometry, are difficult. In the present study, we computationally explored the full pathways for Suc-intermediate formation from Asp residues. The calculations were performed two types of reactant complexes, and all energy minima and TS geometries were optimized using B3LYP density functional methods. As a result, the SI-intermediate formation was divided into three processes, i.e., iminolization, cyclization, and dehydration processes, and the activation energies were calculated to be 26.1 or 28.4 kcal mol−1. These values reproduce the experimental data. The computational results show that abundant water molecules in living organisms are effective catalysts for the Asp-residue isomerization.

Asp 58 modulates lens αA-crystallin oligomer formation and chaperone function.

Senile cataract onset is caused by insolubilization of lens proteins. The lens crystallin protein family correctly orders the formation of homo- or hetero-oligomers in lens fiber cells. Because lens fiber cells do not divide, covalent post-translational modifications, such as isomerization of aspartate residues, accumulate with aging. Although many isomerization sites of αA-crystallin have been reported, their structural and functional contributions have never been identified. In this study, αA-crystallin was extracted from aged human lens and separated into each oligomeric state by size exclusion chromatography and electrophoresis. The novel combination methodology of in-solution/gel tryptic digestion with liquid chromatography equipped with mass spectrometry (LC-MS/MS) was used to evaluate the isomerization of Asp 58. The contributions of isomerization to assembly, solubility, and chaperone functions of αA-crystallin were estimated using a series of mutations of Asp 58 in αA-crystallin. The results indicated that the isomerization of Asp 58 depended on the oligomer size and age of the lens. The substitution of Asp 58 for hydrophobic residues increased αA-crystallin oligomer size and decreased solubility. All substitutions decreased the chaperone function of αA-crystallin for aggregates of bovine βL-crystallin and alcohol dehydrogenase. The data indicated that Asp 58 in αA-crystallin was critical for intermolecular interactions in the lens. Our results also suggested that LC-MS/MS-based isomerization analyses of in-gel-digested products could be useful for investigating the isomerization of Asp residues in oligomeric states. This method could also be used to analyze d/l ratios of amino acid residues in soluble protein aggregates.

A quantitative map of glutathione in the aging human lens

Changes to cellular metabolism often precede changes to other biomolecules that in turn cause tissue pathologies. In the ocular lens, changes in the spatial distribution and concentration of metabolites that are associated with the aging process are implicated in the development of the primary lens pathology, lens cataract. The development of age-related nuclear cataract is characterised by regional changes to protein cross-linking and protein insolubilisation that subsequently results in cataract formation. In the present study, MALDI FT-ICR imaging mass spectrometry was used to map the metabolome of the aging human ocular lens, and a MALDI imaging mass spectrometry method developed to quantify regional changes to the primary lens antioxidant glutathione (reduced). Spatial segmentation of total metabolite signal intensities showed the lens contains distinct metabolic compartments that change with increasing age. Using the developed absolute quantitation method, glutathione concentrations throughout the lens were mapped with up to 100μm spatial resolution, and showed that cortical concentrations (mean=3.7μmol/g tissue in 34yo) were always higher than nuclear concentrations (mean=2.8μmol/g tissue in 34yo). In addition, cortical concentrations decreased slightly with increasing lens age (mean=2.7μmol/g tissue in 74yo), while the decrease in nuclear glutathione concentrations was more pronounced (mean=0.8μmol/g tissue in 74yo). Finally, ceramides were localised to the lens region that was specifically depleted of glutathione. In summary, MALDI FT-ICR imaging mass spectrometry has allowed age-related changes to human lens metabolites to be mapped with higher spatial resolution than previously possible using alternative techniques. In addition, the multiplex capability of MALDI imaging mass spectrometry has allowed the spatial relationships of these lens metabolites to be established and explored. This approach should aid the development of spatially directed therapies to delay the onset of lens cataract.

RNA aptamers targeted for human αA-crystallin do not bind αB-crystallin, and spare the α−crystallin domain

The molecular chaperones, α−crystallins, belong to the small heat shock protein (sHSP) family and prevent the aggregation and insolubilization of client proteins. Studies in vivo have shown that the chaperone activity of the α−crystallins is raised or lowered in various disease states. Therefore, the development of tools to control chaperone activity may provide avenues for therapeutic intervention, as well as enable a molecular understanding of chaperone function. The major human lens α−crystallins, αA- (HAA) and αB- (HAB), share 57% sequence identity and show similar activity towards some clients, but differing activities towards others. Notably, both crystallins contain the “α−crystallin domain” (ACD, the primary client binding site), like all other members of the sHSP family. Here we show that RNA aptamers selected for HAA, in vitro, exhibit specific affinity to HAA but do not bind HAB. Significantly, these aptamers also exclude the ACD. This study thus demonstrates that RNA aptamers against sHSPs can be designed that show high affinity and specificity – yet exclude the primary client binding region – thereby facilitating the development of RNA aptamer-based therapeutic intervention strategies.

Isomerization of aspartyl residues in crystallins and its influence upon cataract

BackgroundAge-related cataracts, which probably form due to insolubilization of lens proteins, can lead to loss of vision. Although the exact reason is unknown, lens protein aggregation may be triggered by increases in PTMs such as d-β-, l-β- and d-α-Asp isomers. These isomers have been observed in aged lens; however, there have been few quantitative and site-specific studies owing to the lack of a quick and precise method for distinguishing between d- and l-Asp in a peptide or protein. Scope of reviewWe describe a new method for detecting peptides containing Asp isomers at individual sites in any protein by using an LC–MS/MS system combined with commercial enzymes that specifically react with different isomers. We also summarize current data on the effect of Asp isomerization on lens crystallins. Major conclusionsThe new technique enabled the analysis of isomers of Asp residues in lens proteins precisely and quickly. An extensive proportion of Asp isomerization was observed at all Asp sites of crystallins in the insoluble fraction of aged lens. In addition, d-amino acid substitutions in crystallin-mimic peptides showed altered structural formation and function. These results indicate that isomerization of Asp residues affects the stability, structure and inter-subunit interaction of lens crystallins, which will induce crystallin aggregation and insolubilization, disrupt the associated functions, and ultimately contribute to the onset of senile cataract formation. General significanceThe mechanism underlying the onset of age-related diseases may involve isomerization, whereby d-amino acids are incorporated in the l-amino acid world of life.This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Accelerated Oxidation Test of Freeze-Dried Pseudomonas Fluorescens BTP1, BB2 and PI9 Strains

Pseudomonas fluorescens strains are very susceptible to damage during freeze-drying and storage. It would be useful to have longer lasting viable powders. Membrane oxidation is one of the factors responsible for damages suffered by P. fluorescens strains during storage. Singlet oxygen (1O2) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. We exposed freeze-dried powders of three strains P. fluorescens (BB2, BTP1 and PI9) to singlet oxygen produced by photosensitisation of Methylene Blue/ light during 2 hours. We observe a significant loss in survival rate, a pronounced oxidation of polyunsaturated fatty acids, protein oxidation (carbonyl groups) and protein insolubilisation. This study enables better predictions of types of packaging suitable for freeze-dried powders of P. fluorescens.

Autolysis of krill protein from North Pacific krill Euphausia pacifica during protein recovery via isoelectric solubilization/precipitation

Autolysis of North Pacific krill protein in acidic and alkaline solubilization during protein recovery via isoelectric solubilization/precipitation was investigated. Pronounced auto-degradation of myosin heavy chain and actin from krill protein was observed at alkaline pH values (pH 9.0–12.0), with maximum autolytic activity at about pH 12.0. Meanwhile, proteolytic activity in krill protein at pH 12.0 was observed at low temperatures, suggesting a possible cause for the autolysis of krill protein during the protein recovery process. Three major proteinases were detected by zymographic analysis of myofibrillar protein, with approximate molecular weights of 26 kDa (KP-1), 18 kDa (KP-2) and 17 kDa (KP-3). KP-3 was active over a pH range from pH 5.0–12.0, suggesting that it may be responsible for the autolysis of krill protein during alkaline-induced protein recovery. Further study on substrate specificity and inhibitory specificity of KP-3 showed that KP-3 is serine type proteinase.

Efficacy of Biodegradable Curcumin Nanoparticles in Delaying Cataract in Diabetic Rat Model

Curcumin, the active principle present in the yellow spice turmeric, has been shown to exhibit various pharmacological actions such as antioxidant, anti-inflammatory, antimicrobial, and anti-carcinogenic activities. Previously we have reported that dietary curcumin delays diabetes-induced cataract in rats. However, low peroral bioavailability is a major limiting factor for the success of clinical utilization of curcumin. In this study, we have administered curcumin encapsulated nanoparticles in streptozotocin (STZ) induced diabetic cataract model. Oral administration of 2 mg/day nanocurcumin was significantly more effective than curcumin in delaying diabetic cataracts in rats. The significant delay in progression of diabetic cataract by nanocurcumin is attributed to its ability to intervene the biochemical pathways of disease progression such as protein insolubilization, polyol pathway, protein glycation, crystallin distribution and oxidative stress. The enhanced performance of nanocurcumin can be attributed probably to its improved oral bioavailability. Together, the results of the present study demonstrate the potential of nanocurcumin in managing diabetic cataract.

In vivo analysis of protein quality control in response to aging using transgenic mice

AbstractPurpose It has been suggested that aging affects the cells ability to protect protein integrity, which is essential for cellular homeostasis. On cell aging there is impaired protein homeostasis, which can lead to protein insolubilization that is toxic to cells. The carboxyl terminus of Hsp70‐interacting protein (CHIP) is an important ubiquitin ligase for protein quality control, and particularly in selective degradation of aggregation prone proteins. The objective of this study was to characterize the transgenic mice that overexpress CHIP/Ubc5 in lens epithelial cells and investigate the role of these proteins in the process that maintains proteins in solution and therefore participates in a number of age‐related diseases, including cataract.Methods To overexpress CHIP, the α‐crystallin promoter was used to drive the expression of CHIP‐Ubc5 in lens epithelial cells of transgenic mice. The CHIP levels of transgenic mice (Tg) were determined with western blotting analysis. Cataract was induced by single subcutaneous injection of sodium selenite and the lens transparency was assessed by slit lamp examination. The soluble and insoluble protein content in all groups was evaluated.Results The data show that the transgenic mice only overexpress the transgene in lens epithelial cells.The slit lamp analysis showed that transgenic mice appears developed less cataract compared with wild type, with concomitantly increase in ratio of the soluble and insoluble proteins.Conclusion Together the data suggest that overexpression of CHIP/Ubc5 in lens epithelial cells could prevent cataract formation by promoting reduction of protein insolubilization.

Chaperone Peptides of α-Crystallin Inhibit Epithelial Cell Apoptosis, Protein Insolubilization, and Opacification in Experimental Cataracts

α-Crystallin is a member of the small heat-shock protein (sHSP) family and consists of two subunits, αA and αB. Both αA- and αB-crystallin act as chaperones and anti-apoptotic proteins. Previous studies have identified the peptide (70)KFVIFLDVKHFSPEDLTVK(88) in αA-crystallin and the peptide (73)DRFSVNLDVKHFSPEELKVK(92) in αB-crystallin as mini-chaperones. In the human lens, lysine 70 (Lys(70)) of αA and Lys(92) of αB (in the mini-chaperone sequences) are acetylated. In this study, we investigated the cellular effects of the unmodified and acetyl mini-chaperones. The αA- and αB-crystallin peptides inhibited stress-induced aggregation of four client proteins, and the αA-acetyl peptide was more effective than the native peptide against three of the client proteins. Both the acetyl and native crystallin peptides inhibited stress-induced apoptosis in two mammalian cell types, and this property was directly related to the inhibition of cytochrome c release from mitochondria and the activity of caspase-3 and -9. In organ-cultured rat lenses, the peptides inhibited calcimycin-induced epithelial cell apoptosis. Intraperitoneal injection of the peptides inhibited cataract development in selenite-treated rats, which was accompanied by inhibition of oxidative stress, protein insolubilization, and caspase activity in the lens. These inhibitory effects were more pronounced for acetyl peptides than native peptides. A scrambled αA-crystallin peptide produced no such effects. The results suggest that the α-crystallin chaperone peptides could be used as therapeutic agents to treat cataracts and diseases in which protein aggregation and apoptosis are contributing factors.

Rutin ameliorates free radical mediated cataract by enhancing the chaperone activity of α-crystallin

Cataract, the leading cause of blindness, is associated with oxidative damage and protein modification in the lens. The present study was carried out to assess the efficacy of rutin on rat-lens crystallins in selenite-induced in-vivo cataract models. Eight-day-old Sprague-Dawley rat pups were grouped as control (G I), experimental (G II) and rutin-treated (G III). The rat pups in G II, and G III received a single subcutaneous injection of sodium selenite (4μg/g body weight) and G I received a single subcutaneous injection of sterile water on the 10th day. The treatment groups (G III) were administered with rutin (1μg/g body weight) respectively from the 8th to 15th day. Cataract was visualized from the 16th day. Lens crystallins (α, β, and γ) were isolated by size exclusion chromatography. Chaperone activity of isolated crystallins was measured by heat, DTT, and oxidation-induced aggregation and refolding assays. Concentration of total protein (soluble and insoluble) and SDS-PAGE analysis of soluble proteins were also done. Treatment with rutin prevented the loss of α crystallin chaperone property, and protein insolubilization prevailed during selenite-induced cataract. These results suggest the therapeutic potential of rutin, a bioflavonoid, against selenite-induced cataract, which has been reported in this paper for the first time. The work assumes significance, as this is a novel approach in modulating the chaperone activity of lens crystallins in selenite-induced cataract by a natural product.

Role of the CipA Scaffoldin Protein in Cellulose Solubilization, as Determined by Targeted Gene Deletion and Complementation in Clostridium thermocellum

The CipA scaffoldin protein plays a key role in the Clostridium thermocellum cellulosome. Previous studies have revealed that mutants deficient in binding or solubilizing cellulose also exhibit reduced expression of CipA. To confirm that CipA is, in fact, necessary for rapid solubilization of crystalline cellulose, the gene was deleted from the chromosome using targeted gene deletion technologies. The CipA deletion mutant exhibited a 100-fold reduction in cellulose solubilization rate, although it was eventually able to solubilize 80% of the 5 g/liter cellulose initially present. The deletion mutant was complemented by a copy of cipA expressed from a replicating plasmid. In this strain, Avicelase activity was restored, although the rate was 2-fold lower than that in the wild type and the duration of the lag phase was increased. The cipA coding sequence is located at the beginning of a gene cluster containing several other genes thought to be responsible for the structural organization of the cellulosome, including olpB, orf2p, and olpA. Tandem mass spectrometry revealed a 10-fold reduction in the expression of olpB, which may explain the lower growth rate. This deletion experiment adds further evidence that CipA plays a key role in cellulose solubilization by C. thermocellum, and it raises interesting questions about the differential roles of the anchor scaffoldin proteins OlpB, Orf2p, and SdbA.

Influence of high pressure homogenization (HPH) on the structural stability of an egg/dairy emulsion

Abstract High pressure homogenization (HPH) is a novel non-thermal preservation technology, which can improve the microbiological quality of products without affecting their stability. The main objective of this paper is to study the influence of different HPH treatments on the structure of a sauce (an egg/dairy emulsion), in order to obtain the higher physicochemical stability. The oil-in-water emulsion was stable up to 100 MPa with the oil droplets surrounded by several layers of natural emulsifiers. Critical pressures, between 150 and 250 MPa, produced a destabilization of the emulsion thus causing a separation of phases. A coalescence phenomenon progressively occurred when pressure increased. This phenomenon was due to the loss of the natural emulsifier barrier. Changes on the electrophoretic pattern were also observed at high pressure levels, showing an insolubilization of proteins. Lipid fraction was observed to be chemically stable after the HPH treatment.