Rheology and synergy of κ-carrageenan/locust bean gum/konjac glucomannan gels

The rheology and melting of mixed polysaccharide gels containing konjac glucomannan (KGM), locust bean gum (LBG) and κ-carrageenan (KC) were studied. Synergy-type peaks in the Young's modulus at optimal mixing ratios were found for both KC/LBG and KC/KGM binary gels at a fixed total polysaccharide content (1:5.5 for LBG:KC and 1:7 for KGM:KC). The Young's modulus peak for KC/KGM was higher than for KC/LBG gels. The same stoichiometric mixing ratios were found when either LBG or KGM was added to KC at a fixed KC concentration, where the Young's modulus increased up to additions at the stoichiometric ratio, but leveled off at higher LBG or KGM additions. Addition of KGM or LBG to the 2-component gels beyond the stoichiometric (optimal) mixing ratio at a fixed total polysaccharide content led to a decrease in the Young's modulus and an increase in the rupture strain and stress in extension, and both trends were stronger for KGM than for LBG.     

Immobilized growing lactic acid bacteria with κ-carrageenan — locust bean gum gel

Summary A cell entrapment process using -carrageenan — locust bean gum gel is presented. Streptococcus thermophilus, Lactobacillus bulgaricus and S. lactis were immobilized in small gel beads (0.5–1.0 mm and 1.0–2.0 mm diameter) and fermentations in bench bioreactors were conducted. Viability of entrapped cells, lactose utilization, lactic acid production and cell release rates were measured during fermentation. The procedure was effective for S. thermophilus, L. bulgaricus and S. lactis, and the viability of these bacteria remained very high throughout entrapment steps and subsequent storage. Bead diameter influenced the fermentation rate: smaller beads (0.5–1.0 mm) permitted an increase in release rates, lactose utilization and acid production by entrapped cells, approximating values attained with free cells.     

Batch fermentations with a mixed culture of lactic acid bacteria immobilized separately in κ-carrageenan locust bean gum gel beads

Summary Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus were immobilized separately in -carrageenan-locust bean gum gel beads. The beads were prepared by a dispersion process in a two-phase system (water in oil) and two ranges of bead diameter selected by sieving (0.5–1.0 mm and 1.0–2.0 mm). Fermentations with the two strains were conducted in bench bioreactors in a supplemented whey permeate medium. Free and entrapped cells (two ranges of bead diameter and two levels of initial bead cell load) were grown in mixed culture, and carbohydrate utilization, acid production and cell growth or cell release rate measured. Fermentation rates were influenced by bead diameter and initial cell load of the beads. Beads with high initial cell density increased fermentation rates compared to low cell density beads or free cells. Smaller diameter beads (0.5–1.0 mm) showed a stable tendency (not statistically significant p _a > 0.05) towards higher cell release rates, lactose utilization, galactose accumulation and lactic acid production than did larger diameter beads (1.0–2.0 mm). Immobilization of S. salivarius subsp. thermophilus and L. delbrueckii subsp. bulgaricus in separate beads did not seem to affect protocooperation during batch fermentation, and allowed for high cell release rates into the medium.     

Characterization and optimization of a new immobilized system of κ-carrageenan through interaction with carob bean gum and polyols

The optimum relationships of κ-carrageenan and carob bean gum were investigated in order to obtain an immobilization system with better compression resistance, trapping capacity, and storage stability, and less syneresis phenomenon, when compared to κ-carrageenan itself. With that objective, different concentrations of polyols (glycerol and propylene glycol) were added, because of their water-retention characterization in the containing system. In this way, an improved system with good compression resistance was obtained: 15 × 10⁻⁴ N/m² in modified κ-carrageenan gel without E. coli cells and 11 × 10⁻⁴ N/m² with Escherichia coli cells. In the modified κ-carrageenan gel, the syneresis phenomenon decreased. The enzymatic activity in the system was 18 U/g, which did not present a change over a storage period of six months.     

The effect of phosphate buffer in the range of pH 5.80–8.07 on jack bean urease activity

The effect of phosphate buffer on the activity of jack bean urease was studied in the range of pH 5.80–8.07. The inhibition constants of phosphate buffer were determined by measuring initial reaction rates at each pH for a series of buffer concentrations at a series of urea concentrations. It was shown that: (1) at pH 5.80–7.49 the buffer is a competitive inhibitor of the enzyme with K_i,buffer increasing from 0.54 mM for pH 5.80 to 362 mM for pH 7.49, (2) the values of pK_i,buffer are pH-dependent exhibiting a slope of −1 at pH 5.80–6.5 and a slope of −2 at pH 6.5–7.49, (3) from pH 7.62 as the pH is further raised the competitive inhibition of urease by the buffer was not observed, (4) the true competitive inhibitor of urease is H₂PO₄⁻ ion, and (5) pH 6.5 and 7.6 correspond to the ionization constants of the active site groups of urease responsible for the inhibitory strength of H₂PO₄⁻ ion.     

Effect of medium and temperature of storage on viability of lactic acid bacteria immobilized in κ-carrageenan-locust bean gum gel beads

Summary The influence of various storage solutions and temperature (4°C and 25°C) on viability ofStreptococcus salivarius subsp.thermophilus andLactobacillusdelbrueckii subsp.bulgaricus entrapped in κ-carrageenan-locust bean gum mixed gel beads was studied. The immobilized strains could be stored at 4°C in all storage solutions studied for at least 14 and 11 days respectively before counts decreased to 10⁵c.f.u./mL, which was considered to be the practical limit for their use as inoculum in a fermentation process. The most effective storage solutions for preserving cell viability at 4°C were NaCl, glycerol and sorbitol solutions forS. thermophilus, and PO₄ buffer and sorbitol solutions forL. bulgaricus. At 25°C,S. thermophilus could be stored for over 14 days in all solutions except glycerol, andL. bulgaricus for 4 days in 10% sorbitol.     

The immune regulation of κ-carrageenan oligosaccharide and its desulfated derivatives on LPS-activated microglial cells

Neurodegenerative disease involves an inflammatory response in the central nervous system characterized by an increase in inflammatory cytokines and activation of microglial cell. To reveal the immune regulation activity of κ-carrageenan oligosaccharides (KOS) on microglia cell activated by LPS and the relationship between the sulfate group content of KOS and its immune regulation activity, KOS was prepared by enzymatic hydrolysis. The degradation products of κ-carrageenan were analyzed by high performance liquid chromatography (HPLC), ESI-TOF-MS and (13)C NMR spectroscopy, and the results indicated that the hydrolyzed products of the κ-carrageenase were κ-neocarrabiose-sulfate, κ-neocarrahexaose-sulfate and κ-neocarraoctaose-sulfate, respectively. Then desulfated derivatives of KOS (DSK) were obtained with DMSO-methanol-pyridine method. The effect of KOS and DSK on the viability of microglia cell activated by LPS was determined with MTT method. Griess assay and ELISA method were used to determine the contents of NO/NO(2-), TNF-α and IL-10 released by activated microglia cell, respectively. The results showed that KOS could inhibit the viability and content of NO, TNF-α and IL-10 released by LPS-activated microglia cell dose dependently. Compared with that of KOS, the inhibiting activity of DSK is weaker. So it could be concluded that KOS could protect microglial cell from being activated by LPS, and there was a positive relationship between the sulfate group content of KOS and its protection function.     

Production of a high activity of an extremely thermostable β-mannanase by the thermophilic eubacterium Rhodothermus marinus, grown on locust bean gum

The production of a highly thermostable mannanase by Rhodothermus marinus was increased 16.5-fold by optimising the concentrations of locust bean gum and yeast extract using central composite designs. The optimised medium and culture conditions yielded mannanase activity at 495 nkat ml–1 (248 nkat mg–1 protein). In addition, -L-arabinofuranosidase, -xylanase, -xylosidase, -glucosidase, -mannosidase, -galactosidase, -galactosidase and endoglucanase activities were detected at 32 nkat ml–1, 30 nkat ml–1, 16 nkat ml–1, 15 nkat ml–1, 0.1 nkat ml–1, 1 nkat ml–1, 0.5 nkat ml–1 and 8 nkat ml–1, respectively. No filter paper cellulase activity could be detected. The optimum pH of the mannanase was 5.0–6.5 and it showed high stability from pH 5 to 10 after 16 h incubation at 50 °C. The enzyme activity was maximum at 85 °C, with half lives of 45.3 h at 85 °C and 4.2 h at 90 °C. This is the first report on the production of such a high activity of extremely thermostable mannanase by an extreme thermophilic bacterium. © Rapid Science Ltd. 1998     

The effect of rice aging on the freeze–thaw stability of rice flour gels

This study investigated the effect of aging rice on the freeze–thaw stability of rice flour gels since repeated freeze–thaw cycles can lead to reduced food quality. A rice grain cultivar called ‘Khoa Dawk Mali 105’ was aged for three different time periods, ranging from 0 to 12 months. Rice gels made from the aged rice were then freeze–thawed for up to 5 cycles. Repeated freeze–thaw cycles lead to an increase in syneresis values and hardness with increasing rice aging. Differential scanning calorimetry showed an increase in the enthalpy of melting of the amylose–lipid complex after 5 freeze–thaw cycles and an increase in peak gelatinization temperature and gelatinization enthalpy with longer rice aging. Moreover, aging length of the rice correlated significantly with a decrease in peak viscosity and breakdown but also an increase in final viscosity and setback. These results demonstrate that aging the rice reduced the freeze–thaw stability of the rice flour gels.     

The effect of cross-links on the mobility of proteins in dodecyl sulphate–polyacrylamide gels

The effect of reduction of intramolecular disulphide bridges on the mobility of proteins in 5% (w/v) polyacrylamide gels in the presence of sodium dodecyl sulphate was investigated. A series of polypeptide polymers, containing up to 68 intramolecular disulphide bridges, was prepared by cross-linking proteins of known structure with glutaraldehyde. These model polypeptides were denatured with heat, sodium dodecyl sulphate and urea, and their mobilities in sodium dodecyl sulphate-polyacrylamide gels compared before and after reduction with dithiothreitol. The mobilities of polypeptides containing no cystine were unaffected by reduction. However, reduction generally decreased the mobilities of polypeptides containing cystine; the extent of this decrease depended on the number of cystine residues originally present in the polypeptide polymer, and on the protein from which the latter was derived. In contrast with their higher oligomers, the monomer of lysozyme and the dimer of ribonuclease increased in mobility after reduction. The reduced polypeptide oligomers formed by reaction with glutaraldehyde were generally found to migrate at a rate significantly faster than was expected from their calculated molecular weights. It was concluded that the use of unreduced proteins and protein aggregates for molecular-weight measurements by the sodium dodecyl sulphate-polyacrylamide-gel method may give erroneous estimates of the molecular weight of any protein being investigated.     

Jack bean α-mannosidase digestion profile of hybrid-type N-glycans: effect of reaction pH on substrate preference

Jack bean α-mannosidase (JBM) is a well-studied plant vacuolar α-mannosidase, and is widely used as a tool for the enzymatic analysis of sugar chains of glycoproteins. In this study, the JBM digestion profile of hybrid-type N-glycans was examined using pyridylamino (PA-) sugar chains. The digestion efficiencies of the PA-labeled hybrid-type N-glycans Manα1,6(Manα1,3)Manα1,6(GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GNM5-PA) and Manα1,6(Manα1,3)Manα1,6(Galβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GalGNM5-PA) were significantly lower than that of the oligomannose-type N-glycan Manα1,6(Manα1,3)Manα1,6Manβ1,4GlcNAcβ1,4GlcNAc-PA (M4-PA), and the trimming pathways of GNM5-PA and GalGNM5-PA were different from that of M4-PA, suggesting a steric hindrance to the JBM activity caused by GlcNAcβ1-2Man(α) residues of the hybrid-type N-glycans. We also found that the substrate preference of JBM for the terminal Manα1-6Man(α) and Manα1-3Man(α) linkages in the hybrid-type N-glycans was altered by the change in reaction pH, suggesting a pH-dependent change in the enzyme-substrate interaction.     

The effect of cathepsin K deficiency on airway development and TGF-β1 degradation

Background

Human immunodeficiency virus (HIV) infected patients are at increased risk for the development of pulmonary arterial hypertension (PAH). Recent reports have demonstrated that HIV associated viral proteins induce reactive oxygen species (ROS) with resultant endothelial cell dysfunction and related vascular injury. In this study, we explored the impact of HIV protein induced oxidative stress on production of hypoxia inducible factor (HIF)-1α and platelet-derived growth factor (PDGF), critical mediators implicated in the pathogenesis of HIV-PAH.

Methods

The lungs from 4-5 months old HIV-1 transgenic (Tg) rats were assessed for the presence of pulmonary vascular remodeling and HIF-1α/PDGF-BB expression in comparison with wild type controls. Human primary pulmonary arterial endothelial cells (HPAEC) were treated with HIV-associated proteins in the presence or absence of pretreatment with antioxidants, for 24 hrs followed by estimation of ROS levels and western blot analysis of HIF-1α or PDGF-BB.

Results

HIV-Tg rats, a model with marked viral protein induced vascular oxidative stress in the absence of active HIV-1 replication demonstrated significant medial thickening of pulmonary vessels and increased right ventricular mass compared to wild-type controls, with increased expression of HIF-1α and PDGF-BB in HIV-Tg rats. The up-regulation of both HIF-1α and PDGF-B chain mRNA in each HIV-Tg rat was directly correlated with an increase in right ventricular/left ventricular+septum ratio. Supporting our in-vivo findings, HPAECs treated with HIV-proteins: Tat and gp120, demonstrated increased ROS and parallel increase of PDGF-BB expression with the maximum induction observed on treatment with R5 type gp-120_CM. Pre-treatment of endothelial cells with antioxidants or transfection of cells with HIF-1α small interfering RNA resulted in abrogation of gp-120_CM mediated induction of PDGF-BB, therefore, confirming that ROS generation and activation of HIF-1α plays critical role in gp120 mediated up-regulation of PDGF-BB.

Conclusion

In summary, these findings indicate that viral protein induced oxidative stress results in HIF-1α dependent up-regulation of PDGF-BB and suggests the possible involvement of this pathway in the development of HIV-PAH.     

The effect of cold storage at 4 °C on the Rooting of chestnut cuttings

This article reports the effect of cold storage at 4 °C from December to April on chestnut cuttings. Rooting was found to increase after four months preceded by treatment with 4000 mg l^-1 IBA. ThePhaseolus test of the biological activity of extracts of the cuttings showed the inhibition exerted by the acid fraction of the fresh (December) cuttings to have been replaced by a rootpromoting effect in cuttings stored for 4 months at low temperature. Cold storage thus seems to favour rooting.     

Enzymatic degradation of hybrid ι-/ν-carrageenan by Alteromonas fortis ι-carrageenase

Hybrid ι-/ν-carrageenan was water-extracted from Eucheuma denticulatum and incubated with Alteromonas fortis ι-carrageenase. The degradation products were then separated by anion-exchange chromatography. The three most abundant fractions of hybrid ι-/ν-carrageenan oligosaccharides were purified and their structures were analyzed by NMR. The smallest hybrid was an octasaccharide with a ι-ι-ν-ι structure. The second fraction was composed of two decasaccharides with ι-ι-ι-ν-ι and ι-[ι/ν]-ι-ι structures. The third fraction was a mixture of dodecasaccharides which contained at least a ι-ι-ι-ι-ν-ι oligosaccharide. The carbon and proton NMR spectra of the octasaccharides were completely assigned, thereby completely attributing the ν-carrabiose moiety for the first time.     

The effect of context on the folding of β-hairpins

Small β-hairpin peptides have been widely used as models for the folding of β-sheets. But how applicable is the folding of such models to β-structure in larger proteins with conventional hydrophobic cores? Here we present multiple unfolding simulations of three such proteins that contain the WW domain double hairpin β-sheet motif: cold shock protein A (CspA), cold shock protein B (CspB) and glucose permease IIA domain. We compare the behavior of the free motif in solution and in the context of proteins of different size and architecture. Both Csp proteins lost contacts between the double-hairpin motif and the protein core as the first step of unfolding and proceeded to unfold with loss of the third β-strand, similar to the isolated WW domain. The glucose permease IIA domain is a larger protein and the contacts between the motif and the core were not lost as quickly. Instead the unfolding pathway of glucose permease IIA followed a different pathway with β1 pulling away from the sheet first. Interestingly, when the double hairpin motif was excised from the glucose permease IIA domain and simulated in isolation in water it unfolded by the same pathway as the WW domain, indicating that it is tertiary interactions with the protein that alter the motif’s unfolding not a sequence dependent effect on its intrinsic unfolding behavior. With respect to the unfolding of the hairpins, there was no consistent order to the loss of hydrogen bonds between the β-strands in the hairpins in any of the systems. Our results show that while the folding behavior of the isolated WW domain is generally consistent with the double hairpin motif’s behavior in the cold shock proteins, it is not the case for the glucose permease IIA domain. So, one must be cautious in extrapolating findings from model systems to larger more complicated proteins where tertiary interactions can overwhelm intrinsic behavior.     

The binding of inhibitors to α-chymotrypsin at alkaline pH

1. The binding of the competitive inhibitor N-acetyl-d-tryptophan amide to alpha-chymotrypsin has now been studied at pH values up to 10.6, by the technique of equilibrium dialysis. 2. This binding depends on the ionization of a group on the free enzyme with apparent pK(a) 9.3 at 5 degrees . 3. This group is tentatively identified as that responsible for an enzyme conformation change at high pH values, on which the catalytic activity of the enzyme also depends.     

The effect of pH on α-methyl-d-glucoside accumulation by rat kidney cortex slices

The kinetics of α-methyl-d-glucoside accumulation by rat kidney cortex slices under conditions of varying extracellular pH are compared with values obtained at pH 7.4. At pH below 7.4 there is a diminished initial uptake and reduced influx of the sugar which results in a decrease in the steady-state intracellular pool. This was associated with a decrease in the $\text{V}$ of the entry process without affecting the apparent $\text{K}{}_{\mathrm{m}}$ of transport. At pH 8 there is no change in the rate of glucoside entry. The efflux of the glucoside, however, is impaired and the steady-state concentration gradient becomes greater than that observed at pH 7.4.     

The effects of γ-interferon on human peripheral blood monocyte/macrophage-mediated bone particle degradation

γ-Interferon (IFN-γ) has recently been demonstrated to inhibit the ability of mononuclcar phagocytes to degrade bone particles. We have further addressed the specificity, potency and mechanism of this activity using human recombinant IFN-γ. Adherent peripheral blood mononuclear leukocytes from normal human volunteers were cultured with washed, sieved (⩽75 μm) ⁴⁵Ca-labelled rat bone particles for 3 days, after which bone particle degradation (7.1 ± 1.6%, n = 11) was calculated from the fraction of⁴⁵Ca released into the medium. As little as 5 U/ml IFN-γ significantly suppressed bone particle degradation and 50 U/ml was associated with consistent marked suppression (74.0 ± 3.5% inhibition, P < 0.001, n = 11). IFN-γ was not suppressive if added to cells 24 h or more after exposure to bone particles. Addition of indomethacin (10 μM) did not reverse the effect of IFN-γ, suggesting that it was not prostaglandin-mediated. In addition, 1,25(OH)₂D₃ (10 nM) did not remove the inhibitor, effect of IFN-γ.Contact of mononuclear phagocytes with bone particles and secretion of soluble factors from these cells have both been demonstrated to play a role in their ability to degrade bone particles. IFN-γ (50 U/ml) inhibited monocyte/macrophage interaction with another unopsonized surface, i.e., one μm fluorescent latex particles, decreasing the number of internalized particles from 12.6 ± 2.9 per cell to 5.9 ± 1.4 per cell (P < 0.01, n = 15), as measured using flow cytometry. However, binding of bone particles by the cells was not diminished by IFN-γ. Exogenous α-imerferon and human recombinant IL-1β, TNF-α, and lymphotoxin did not alter bone particle degradation. In addition, endogenous IL-1β release from human monocyte/macrophages exposed to bone particles was negligible and unaffected by IFN-γ.We conclude that IFN-γ is a potent and specific inhibitor of monocyte/macrophage-mediated bone particle degradation, and that this activity does not appear to be due to effects on the ability of monocytes to bind bone particles or to release IL-1 in response to the particles