The effect of modification and fragmentation of α-lactalbumin on lactose and lactosamine synthase reactions

In the presence of the modifier protein -lactalbumin, bovine milk galactosyltransferase transfers galactose to glucose forming lactose instead of transferring toN-acetylglucosamine formingN-acetyllactosamine. At low concentrations of -lact-albumin, the lactosamine synthase activity is stimulated by -lactalbumin and decreases when the lactose synthase activity develops along a sigmoidal curve. The observation suggests that different interactions between -lactalbumin and enzyme were responsible for the modulating effect of the -lactalbumin in the lactose and lactosamine synthase reactions.To study the nature of the protein-protein interactions, -lactalbumin was both modified and cleaved chemically. Reduction and alkylation with iodoacetic acid, iodoacetamide or 4-vinylpyridine abolished the ability of the -lactalbumin to induce lactose synthase activity but stimulated lactosamine synthase activity 7-to 12-fold.A peptide fragment corresponding to residues 26–60 of -lactalbumin isolated from a 2-(2-nitrophenylsulphenyl)-3-methyl-3-bromo-indolene (BNPS-skatole) fragmentation of the molecule was active in the lactosamine but not lactose synthase reaction. We concluded that, whereas lactose synthase required -lactalbumin, in the native conformation, lactosamine synthase activity was stimulated by a linear sequence of amino acids in peptide 26–60.Abbreviations MES 4-N-morpholinoethanesulfonic acid - TRIS 2-amino-2-(hydroxymethyl)-1,3-propanediol - UDP-Gal uridinediphosphogalactose - BNPS-skatole 2-(2-nitrophenylsulphenyl)-3-methyl-3-bromo-indolene - EDTA ethylene diamine tetra acetic acid     

The effect of α-aminoisobutyric acid on wood decay and wood spoilage fungi

The amino acid analogue α-aminoisobutyric acid (AIB) decreased linear extension growth in fifteen out of sixteen wood decay and wood spoilage fungi. In Serpula lacrimans inhibition of extension growth by AIB was accompanied by an increase in the frequency with which the hyphae of the fungus initiated branches. AIB was shown to have a preservative effect against Lentinus lepideus, Serpula lacrimans and Pleurotus ostreatus when wood blocks were impregnated with this chemical prior to challenge by cultures of these fungi. The effectiveness of this compound in limiting growth in a large number of different fungi suggests that competitive inhibitors of nitrogen uptake and metabolism could be used to control fungi which decay wood and similar materials, and may also have wider applications.     

The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin

N^ε-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N^ε-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N^ε-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N^ε-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac₂O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N^ε-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function.     

The effect of hexapole and vertical fields on α-particle confinement in heliotron configurations

Collisionless confinement of monoenergetic α particles in three-dimensional magnetic fields produced by the magnetic coils of the Large Helical Device is calculated. It is found that the inward shift of the magnetic axis due to the vertical field improves the α-particle confinement. In contrast to the vertical field, both large positive and negative hexapole fields do not improve the confinement. The study of the β effect and Mercier criterion calculations for different hexapole fields are also presented.     

The effect of α-tocopherol, α- and γ-tocotrienols on amyloid-β aggregation and disaggregation in vitro

One of the neuropathological hallmarks of Alzheimer's disease (AD)—causing neurodegeneration and consequent memory deterioration, and eventually, cognitive decline—is amyloid-β (Aβ) aggregation forming amyloid plaques. Our previous study showed the potential of a tocotrienol-rich fraction—a mixture of naturally occurring of vitamin E analogs—to inhibit Aβ aggregation and restore cognitive function in an AD mouse model. The current study examined the effect of three vitamin E analogs—α-tocopherol (α-TOC), α-tocotrienol (α-T3), and γ-tocotrienol (γ-T3)—on Aβ aggregation, disaggregation, and oligomerization in vitro. Thioflavin T (ThT) assay showed α-T3 reduced Aβ aggregation at 10 μM concentration. Furthermore, both α-T3 and γ-T3 demonstrated Aβ disaggregation, as shown by the reduction of ThT fluorescence. However, α-TOC showed no significant effect. We confirmed the results for ThT assays with scanning electron microscopy imaging. Further investigation in photo-induced cross-linking of unmodified protein assay indicated a reduction in Aβ oligomerization by γ-T3. The present study thus revealed the individual effect of each tocotrienol analog in reducing Aβ aggregation and oligomerization as well as disaggregating preformed fibrils.     

The effect of vitamin A deficiency and dietary α-tocopherol on the stability of rat-liver lysosomes

1. Vitamin A-deficient rats and pair-fed controls were maintained on either normal or raised amounts of dietary alpha-tocopherol. 2. Their livers were fractionated and ;free' and ;total' lysosomal phosphatase were determined in the various fractions. The rate of release of this enzyme was determined in the mitochondria-lysosome-rich fraction during incubation at pH5 and 37 degrees . 3. The deficient livers showed increased enzymic activity. 4. Prolonged incubation caused more rapid enzyme release from the mitochondria-lysosome-rich fraction of the vitamin A-deficient rats receiving the normal amount of dietary alpha-tocopherol than from the equivalent fraction of their pair-fed controls receiving vitamin A. Raised dietary alpha-tocopherol reversed this phenomenon.     

The effect of monensin on intracellular transport and secretion of α-amylase isoenzymes in barley aleurone

The effect of monensin on the secretion of -amylase and other enzymes from the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya) was studied by electrophoresis followed by fluorography and by pulse-chase and organelle-isolation experiments. Monensin markedly inhibits the secretion, but not the synthesis, of -amylase, acid phosphatase, and at least four other proteins from the aleurone layer. Monensin treatment causes -amylase to accumulate within the protoplast, but its effect on the different -amylase isoenzymes is not equal. The accumulation of isoenzyme 2 is not influenced by monensin while isoenzymes 1, 3 and 4 are not secreted but rather accumulate in the cell when monensin is included in the incubation medium. The -amylase and acid-phosphatase activities which accumulate within the aleurone cells following treatment with monensin are localized in an organelle having a buoyant density greater than that of endoplasmic reticulum and less than that of mitochondria. In pulse-chase experiments with [³⁵S]methionine, labelled proteins accumulate in this organelle in the presence of monensin and do not appear in the incubation medium. We conclude that monensin inhibits the secretion of proteins from the barley aleurone layer by influencing their intracellular transport.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - SDS-PAGE sodium dodecyl-sulfate polyacrylamide-gel electrophoresis     

The effect of birthplace on heat tolerance and mortality in Milan, Italy, 1980–1989

The temperature–mortality relationship follows a well-known J-V shaped pattern with mortality excesses recorded at cold and hot temperatures, and minimum at some optimal value, referred as Minimum Mortality Temperature (MMT). As the MMT, which is used to measure the population heat-tolerance, is higher for people living in warmer places, it has been argued that populations will adapt to temperature changes. We tested this notion by taking advantage of a huge migratory flow that occurred in Italy during the 1950s, when a large number of unemployed people moved from the southern to the industrializing north-western regions. We have analyzed mortality–temperature relationships in Milan residents, split by groups identified by area of birth. In order to obtain estimates of the temperature-related risks, log-linear models have been used to fit daily death count data as a function of different explanatory variables. Results suggest that mortality risks differ by birthplace, regardless of the place of residence, namely heat tolerance in adult life could be modulated by outdoor temperature experienced early in life. This indicates that no complete adaptation might occur with rising external environmental temperatures.     

Inhibition of staphylococcal α-toxin. The effect of aromatic polysulphonic acids on the lethal effect of α-toxin in mice

1. Certain aromatic polysulphonic acids, previously tested for inhibition of the haemolytic activity of staphylococcal α-toxin, together with some additional related compounds, were tested as possible inhibitors of α-toxin in mice. 2. Compounds that inhibited the haemolytic activity of α-toxin at concentrations of 0·16mm or less [compounds (I), (II), (IV), (V), (VII) and (VIII)] were found to inhibit the lethal effect of α-toxin. 3. With the exception of compound (VIII), amounts of 1mg. were required to inhibit 4 LD₅₀ of toxin when the test compounds were premixed with α-toxin before injection; comparable inhibition with 0·3mg. of compound (VIII) was achieved without prolonged premixing. 4. Mixtures of α-toxin and compounds (I) and (II) containing an excess of test compound showed markedly diminished inhibitory activities. 5. The `half-molecule' analogues of group 1 [compounds (III) and (XVIII)] were non-inhibitory. 6. Compounds (I)–(V), when administered separately from α-toxin by the same route (intraperitoneal), were active only when injected almost simultaneously with toxin, whereas compounds (VII) and (VIII) were strikingly inhibitory when injected 15min. before or after the toxin. 7. Compound (VIII) failed to inhibit the lethal effect of α-toxin when injected by a different route (intravenous).     

More diversity,less tolerance? The effect of type of cultural diversity on the erosion of tolerance in Swiss municipalities

Immigration and the ways in which host societies receive newcomers pose challenges for modern civil societies. This article contributes to the ongoing discussion about how ethnic diversity influences tolerance towards immigrants. Compared to previous studies, we analyse tolerance as a sequential concept in order to uncover the effects of contextual diversity on attitudes towards immigrants and the granting of certain rights to this group. Moreover, we distinguish different shares of ethnic groups based on their ethnic and cultural origins both on the independent and dependent variable. The analysis relies on a subnational survey of sixty municipalities in Switzerland, revealing that only certain ethnic groups are seen as an economic and cultural threat.     

Effect of prolyl-leucyl-glycinamide and α-melanocyte-stimulating hormone on levorphanol-induced analgesia,tolerance and dependence

Prolyl-leucyl-glycinamide (PLG) at a low dose (10 ng/mouse) administered by an intracerebroventricular (i.c.v.) injection did not affect levorphanol analgesia, but PLG at higher doses (10 and 100 μg/mouse) and α-melanocyte-stimulating hormone (α-MSH) (10 ng/ mouse) antagonized levorphanol analgesia. Development of levorphanol tolerance was facilitated by 10 ng/mouse of PLG, unaffected by 10 μg/mouse of PLG, but antagonized by 100 μg/mouse of PLG and 10 ng/mouse of α-MSH. The effect of PLG on levorphanol dependence was assessed by changes in body weight and temperature during naloxone-induced withdrawal. PLG (10 ng/mouse) facilitated the development of levorphanol dependence, but 10 μg/mouse of PLG had no effect. PLG (100 μg/mouse) antagonized development of levorphanol dependence. PLG at doses of 10 and 100 μg/mouse precipitated withdrawal in levorphanol-dependent mice. α-MSH (10 ng/mouse) antagonized development of levorphanol dependence as evidenced by an increase in the ED50 of naloxone required to induce withdrawal jumping. These results indicate that PLG and α-MSH affected levorphanol-induced analgesia, tolerance and dependence in a qualitatively similar manner to their effect on morphine-induced analgesia, tolerance and dependence.     

The Effect of Time-of-Day and Sympathetic α1-Blockade on Orthostatic Tolerance

Tolerance time to a standardized orthostatic stressor is markedly reduced in normotensive individuals in the morning. However, the physiological mechanisms that underpin this phenomenon are unknown. The purpose of this study was to examine the role of α₁-adrenergic activity on orthostatic tolerance and associated cardiorespiratory and cerebrovascular responses, and to determine whether its endogenous modulation is important in the diurnal variation of orthostatic tolerance. In a four-trial, randomized placebo-controlled crossover experiment, 12 normotensive volunteers (aged 25?±?1 yrs; mean?±?SE) completed a 60° head-upward tilt (HUT; 15?min or until onset of presyncope) at 06:00 and 16:00?h, 90?min after the administration of either α₁-blockade (prazosin, 1?mg/20?kg body weight) or placebo. Continuous beat-to-beat measurements of middle cerebral blood flow velocity (transcranial Doppler), blood pressure (Finometer), heart rate, stroke volume, cardiac output, and end-tidal carbon dioxide were obtained. Independent of time-of-day, α₁-blockade markedly reduced the ability to tolerate a 15-min 60° HUT; tolerance time was 229% shorter compared with the placebo condition (p?≤?.0001). Moreover, a marked diurnal variation in orthostatic tolerance was evident following α₁-adrenergic blockade; e.g., tolerance time in the morning (176?±?30 s) was lower than in the afternoon (354?±?75 s; p?=?.04). These findings highlight an important role of α₁-sympathetic vasoconstrictor activity in acutely regulating blood pressure and offsetting syncope, especially in the early morning. (Author correspondence: Nia.Lewis@ubc.ca)     

The effect of mechanical load on integrin subunits α5 and β1 in chondrocytes from mature and immature cartilage explants

Articular cartilage is subjected to cyclic compressive stresses during joint loading. There is increasing experimental evidence that this loading is essential for the chondrocytes to maintain the functionality of the cartilage extracellular matrix (ECM) and that members of the integrin family of transmembrane receptors may play an important role in signal mechanotransduction between the ECM and chondrocytes. Of particular interest are the integrin subunits 5 and 1, which are known to form the receptor for fibronectin, an important ECM protein, and to be involved in mechanotransduction as well as in the regulation of cytokine production. In this study, we measured the amounts of the integrin subunits 5 and 1 in chondrocytes from young (immature) and adult (mature) bovine articular cartilage explants which were subjected to a continuously applied cyclic compressive stress of 1 MPa for 6 and 24 h. The integrin content per chondrocyte was measured immediately after load cessation by flow cytometry following matrix digestion to release the cells. We found that a mechanical stress induced an increase in the number of integrin subunit 5 in immature and mature cartilage but not in the integrin subunit 1 content. The integrin contents were greatest after 6 h of loading and returned to control levels after 24 h of unloading. The results of this study supply further experimental evidence that chondrocytes respond to changes in their mechanical environment and that the integrin 51 may act as a mechanical signal transducer between the chondrocyte and the ECM for the modulation of cellular physiology.This work was supported by NIH grant AR45748 to PAT and the HSS MacArthur Cartilage Fund.     

Inhibitory effect of components from Streptomyces species on α-glucosidase and α-amilase of different origin

The search for the effective and safe α-glucosidase and α-amylase inhibitors from Actinomycetaceae being antidiabetic agents is actual problem. Twenty one Streptomyces spp. of soil samples collected from different places of China were screened for the ability to produce this kind of inhibitory activities. Fermentation broth of isolated strains had absorbance between 350–190 nm. The Streptomyces strains PW003, ZG636, and ZG731 were characterized by special absorption at 280, 275, and 400 nm, respectively. Ten of the collected actinomycete strains had the ability to inhibit α-glucosidase or/and α-amylase and the fermentation broth of the same strain had inhibitory activity varied greatly depending on the enzyme source. In the process to screen the leading compounds used as antidiabetic agents, human α-glucosidase and α-amylase were revealed as the best used in trail compared with the same enzymes from other sources. Active α-glucosidase inhibitor was isolated from Streptomyces strain PW638 fermentation broth and identified as acarviostatin I03 by MS and NMR spectrometry. Its IC₅₀ value was 1.25 and 12.23 μg/ml against human intestinal N-terminal maltase-glucoamylase and human pancreatic α-amylase, respectively.     

The effect of various secretagogues on accumulation of cyclic AMP and secretion of α-amylase from rat exocrine pancreas

The relationship between accumulation of cyclic AMP and the secretion of α-amylase was investigated in the rat pancreas $\text{in vitro}$. Theophylline and secretin induced an increase in tissue cyclic AMP levels, however, only secretin stimulated secretion of α-amylase. Pancreozymin caused a release of α-amylase and had a biphasic effect on nucleotide levels — stimulation followed by inhibition. Carbachol, which induced a secretory response in the rat pancreas, reduced tissue levels of the cyclic nucleotide.     

The effect of OPA1 on mitochondrial Ca²⁺ signaling

The dynamin-related GTPase protein OPA1, localized in the intermembrane space and tethered to the inner membrane of mitochondria, participates in the fusion of these organelles. Its mutation is the most prevalent cause of Autosomal Dominant Optic Atrophy. OPA1 controls the diameter of the junctions between the boundary part of the inner membrane and the membrane of cristae and reduces the diffusibility of cytochrome c through these junctions. We postulated that if significant Ca²⁺ uptake into the matrix occurs from the lumen of the cristae, reduced expression of OPA1 would increase the access of Ca²⁺ to the transporters in the crista membrane and thus would enhance Ca²⁺ uptake. In intact H295R adrenocortical and HeLa cells cytosolic Ca²⁺ signals evoked with K⁺ and histamine, respectively, were transferred into the mitochondria. The rate and amplitude of mitochondrial [Ca²⁺] rise (followed with confocal laser scanning microscopy and FRET measurements with fluorescent wide-field microscopy) were increased after knockdown of OPA1, as compared with cells transfected with control RNA or mitofusin1 siRNA. Ca²⁺ uptake was enhanced despite reduced mitochondrial membrane potential. In permeabilized cells the rate of Ca²⁺ uptake by depolarized mitochondria was also increased in OPA1-silenced cells. The participation of Na⁺/Ca²⁺ and Ca²⁺/H⁺ antiporters in this transport process is indicated by pharmacological data. Altogether, our observations reveal the significance of OPA1 in the control of mitochondrial Ca²⁺ metabolism.     

The effect of dietary l-carnitine supplementation on cold tolerance and growth of the ornamental cichlid fish Pelvicachromis pulcher—preliminary results

The effect of dietary l-carnitine supplementation on cold tolerance, growth and survival of the ornamental cichlid Pelvicachromis pulcher was tested under laboratory conditions. Fish were reared for a period of 82 days on a diet containing different levels of added l-carnitine: 0, 500 (3.1 mmol), 1000 (6.2 mmol) and 2000 (12.4 mmol) mg/kg. At the end of the growth period the fish were exposed to a cold shock. Fish which received dietary l-carnitine supplementation (at all tested levels) exhibited significantly better survival rates following exposure to a cold shock, and readapted quicker to normal temperatures compared to the control group which had no l-carnitine added to their diet. The addition of l-carnitine to the diet at a level of 1000 mg/kg seems to yield the best protection against exposure to cold shock.Growth differences among the treated fish were not significant, although the fish which received 500 mg/kg supplementation exhibited slightly better growth.     

The effect of α-phenyl-N-t-butylnitrone on ionizing radiation-induced apoptosis in U937 cells

Ionizing radiation induces the production of reactive oxygen species (ROS), which play an important causative role in apoptotic cell death. α-Phenyl-N-t-butylnitrone (PBN) is one of the most widely used spin-trapping compounds for investigating the existence of free radicals in biological systems. We investigated the effects of PBN on ionizing radiation-induced apoptosis in U937 cells. Upon exposure to 2 Gy of γ-irradiation, there was a distinct difference between the control cells and the cells pre-treated with 2 mM PBN for 2 h in regard to apoptotic parameters, cellular redox status, mitochondria function and oxidative damage to cells. PBN effectively suppressed morphological evidence of apoptosis and DNA fragmentation in U937 cells exposed to ionizing radiation. The [GSSG]/[GSH+GSSG] ratio and the generation of intracellular ROS were higher and the [NADPH]/[NADP⁺+NADPH] ratio was lower in control cells compared to PBN-treated cells. The ionizing radiation-induced mitochondrial damage reflected by the altered mitochondrial permeability transition, the increase in the accumulation of ROS, and the reduction of ATP production were significantly higher in control cells compared to PBN-treated cells. PBN pre-treated cells showed significant inhibition of apoptotic features such as activation of caspase-3, up-regulation of Bax and p53, and down-regulation of Bcl-2 compared to control cells upon exposure to ionizing radiation. This study indicates that PBN may play an important role in regulating the apoptosis induced by ionizing radiation presumably through scavenging of ROS.