A model for cleavage ofO-glycosidic bonds in glycoproteins

The present work investigated the possibility of cleavage of -linkages between mannose or galactose and serine/threonine residues by -mannosidase and -galactosidase. The study was carried out initially with model synthetic compounds imitating theO-glycosidic bond in glycoproteins, and further with glucoamylase. It was shown that -mannosidase and -galactosidase can hydrolyse these linkages after proteolytic digestion of glucosamylase.     

Rate of free-radical oxidation of C18 diene and triene fatty acids in aqueous micellar solutions and effectiveness of beta-carotene as an inhibitor of their oxidation

The rate of accumulation of conjugated dienes of polyunsaturated fatty acids was measured during free-radical oxidation of linoleic acid (18:2n-6, LA), -linolenic acid (18:3n-3, -LNA), and -linolenic acid (18:3n-6, -LNA) initiated by 2,2"-azo-bis-(2-amidinopropane) hydrochloride in aqueous micellar solutions of sodium dodecyl sulfate and sodium cholate. It was shown that, unlike homogeneous solutions, the oxidative stability of PUFAs in aqueous dispersions increased with an increase in the extent of unsaturation. The rate of LA oxidation was more than tenfold greater than that of - and -LNA. The antioxidant activity of -carotene, in contrast to homogeneous solutions, in both micellar systems studied depended on the degree of PUFA unsaturation. We found that 5 M -carotene effectively inhibited the LA oxidation (almost by 90%), whereas the oxidation of -LNA and -LNA was not inhibited by -carotene even at much greater concentration (30 M). The paradoxical discrepancy between the extent of unsaturation and the PUFA oxidation rate, as well as a decrease in the efficiency of -carotene-dependent inhibition of oxidation of more polyunsaturated fatty acids in reactions conducted in aqueous dispersions is consistent with the model according to which the peroxyl radicals of LA and fatty acids with the doublebond number greater than two exhibit different polarity.     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

Two forms of α-glucosidase from sugar-beet seeds

Two forms of -glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from sugarbeet (Beta vulgaris L.) seeds by a procedure including fractionation with ammonium sulfate and ethanol, carboxymethyl-cellulose column chromatography, and preparative disc gel electrophoresis. The two enzymes were homogeneous by polyacrylamide disc gel electrophoresis. Their molecular weights were 98,000 (I) and 60,000 (II). -Glucosidase I readily hydrolyzed maltose, isomaltose, kojibiose, maltotriose, panose, amylose, soluble starch, amylopectin and glycogen. -Glucosidase II also hydrolyzed maltose, kojibiose and maltotriose but hydrolyzed the other substrates only very weakly or not at all. -Glucosidase I hydrolyzed soluble starch at a faster rate than maltose. It produced isomaltose and panose as the main -glucosyltransfer products from maltose, whereas maltotriose was the main product of -glucosidase II. -Glucosidase I hydrolyzed amylose liberating -glucose. The neutral-sugar content was calculated to be 2.7% for -glucosidase I and 8.8% for -glucosidase II. The main neutral sugar was mannose in -glucosidase I, and glucose in -glucosidase II.     

α-Subunit Composition of Nicotinic Cholinoreceptors of Neurons of the Inferior Mesenteric Ganglion of the Guinea Pig: an Electrophysiological Study

We studied the subunit composition of nicotinic cholinoreceptors (nChR) of neurons of the inferior mesenteric ganglion (IMG) of the guinea pig using antibodies against the ₃, ₄, ₅, and ₇ nChR subunits and a standard technique of intracellular recording. Application of the ₃ subunit-specific antibodies resulted in a decrease in the EPSP amplitude by 29.7 ± 1.8%, on average, in 17 of 20 studied neurons. The ₄ subunit-specific antibodies evoked no changes in the amplitude of EPSP recorded from IMG neurons (n = 10). Effects of the ₅ subunit-specific antibodies were studied in 20 neurons, where the EPSP amplitude dropped after application of these antibodies by 40.0 ± 1.8%, on average. Superfusion of the neurons with a solution containing the ₃ and ₅ subunit-specific antibodies completely suppressed synaptic responses (n = 3). The ₇ subunit-specific antibodies provided an increase in the EPSP amplitude in 13 of 15 studied IMG neurons (by 37 ± 6%, on average); this fact allows us to suppose that ₇-containing nChR are present in the IMG neurons and are indirectly involved in the processes of synaptic transmission. Application of the antibodies evoked no significant shifts in the membrane potential in IMG neurons. Our findings demonstrate that synaptic transmission through the guinea pig IMG is realized mostly with the involvement of the ₃- and ₅-containing nChR; the ₄-containing receptors are not engaged in this process.     

Evaluation of hydrophobicity versus chaperonelike activity of bovine alphaA- and alphaB-crystallin

Calf lens A-crystallin isolated by reversed-phase HPLC demonstrates a slightly more hydrophobic profile than B-crystallin. Fluorescent probes in addition to bis-ANS, like cis-parinaric acid (PA) and pyrene, show higher quantum yields or Ham ratios when bound to A-crystallin than to B-crystallin at room temperature. Bis-ANS binding to both A- and B-crystallin decreases with increase in temperature. At room temperature, the chaperone-like activity of A-crystallin is lower than that of B-crystallin whereas at higher temperatures, A-crystallin shows significantly higher protection against aggregation of substrate proteins compared to B-crystallin. Therefore, calf lens A-crystallin is more hydrophobic than B-crystallin and chaperone-like activity of -crystallin subunits is not quantitatively related to their hydrophobicity.     

Ontogeny of alpha-crystallin subunits in the lens of human and rat embryos

The distribution of A- and B-crystallin in the developing lens of human (Carnegie stages 13 to 23) and rat embryos (embryonic days E11 to 18) was examined immunohistochemically. In a human embryo at stage 13, the lens placode was already immunoreactive to B-crystallin, but not to A-crystallin. At stage 15, the lens vesicle was intensely immunoreactive both to A- and B-crystallin. From stages 16 to 23, the lens epithelial cells and fiber cells were immunoreactive to A- and B-crystallin. In rat embryos, A-crystallin appeared in the lens pit at E12, and B-crystallin appeared in the elongating lens fiber cells at E14. From E15 to E18, the lens epithelial cells and fiber cells were immunoreactive to A-crystallin. The lens fiber cells were also immunoreactive to B-crystallin, but the epithelial cells were not. These findings suggest that B-crystallin appears earlier than A-crystallin in the human lens, but at a later period than A-crystallin in the rat lens. B-Crystallin was not detected in the epithelial cells of the rat lens, but was perisistently present in the epithelial cells of the human lens.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

TNF alpha is required for hypoxia-mediated right ventricular hypertrophy

Hypoxia has been shown to activate the pleiotropic cytokine TNF in the lung. TNF in turn, is known to induce pulmonary vasoconstriction. Additional effects of this cytokine in hypoxia mediated cardiopulmonary remodeling are poorly understood. To further evaluate the role of TNF in chronic hypoxia we exposed TNF null (TNF–/–) and wild-type mice to three weeks of hypobaric hypoxia (10% O₂). Equivalent erythocytosis (Hematocrit increased by 40%) developed in both genetic backgrounds. In contrast, right ventricular systolic pressure increased in response to three weeks of hypoxia in the wild-type mice ( 75%), yet was unaltered in the TNF–/– mice. Concomitantly right ventricular hypertrophy was attenuated in the TNF–/– mice (35 ± 5% increase) when compared to wild-type mice (124 ± 6% increase p < 0.001, n 20). Interestingly in both strains the lung wet weights increased to a similar degree in response to hypoxia. In conclusion, our data demonstrate that TNF is an integral autocoid in chronic hypoxia mediated right ventricular hypertrophy. Moreover, additional components of cardiopulmonary remodeling may be regulated by TNF signaling as suggested by the negligible right ventricular systolic pressure response to hypoxia in the absence of TNF.     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

Fungal thermostable α-dialkylamino acid aminotransferase: occurrence,purification and characterization

-Dialkylamino acid aminotransferase was found in various fungi; this is the first evidence for the occurrence of the enzyme in eukaryotes. The enzyme was purified from Fusarium solani and shown to be composed of four subunits with an identical molecular weight of 42,000. -Aminoisobutyrate and cycloleucine served as amino donors, and pyruvate, -ketobutyrate, -ketovalerate, -ketoisovalerate, and glyoxylate as amino acceptors. The K _m values for -aminoisobutyrate and -ketobutyrate were 28 and 0.3 mM, respectively. -Ketobutyrate inhibited the enzyme noncompetitively with -aminoisobutyrate, and showed K _i value of 8 mM. The significant inhibitory effect of l-cycloserine was observed, but d-cycloserine did not inhibit the enzyme. The pH and temperature optima for transamination of -aminoisobutyrate with pyruvate were about 8.0 and 60°C, respectively. Despite the production of this enzyme by the mesophile, the enzyme was thermostable; it retained its full activity upon heating at 60°C for 30 min.Abbreviations ACPC 1-aminocyclopropane-1-carboxylic acid - AIB -aminoisobutyrate - PLP pyridoxal 5-phosphate     

Organization of α-chain genes among Hb G-Philadelphia heterozygotes in association with Hb S, β-thalassemia,and α-thalassemia-2

The percentages of the -chain variant Hb G-Philadelphia (Hb G) or ₂ 68 AsnLys₂ were evaluated in 84 adult and 18 newborn heterozygotes. These included members of three families who were studied in more detail by nucleic acid hybridization techniques. The adult heterozygotes fell in two categories, one with a higher proportion of Hb G [46.5±1.0% (SD), N=21] and another with lower values (33.9±3.4%, N=63). Among the newborn heterozygotes, two babies fell in the category with the higher proportion of Hb G while 16 babies gave values between 25 and 34%. Studies of -chain gene organization on the parents of one neonate with a Hb G level of 27% at birth and 37% at 8 months excluded the presence of chromosomes with triplicated -chain genes which could lead to the ^0G/ genotype. Rather, these studies on five Hb G heterozygotes from three families confirmed the linkage between Hb G and a specific type of -thalassemia-2 associated with the presence of a 16-kbp Bgl II fragment which most probably carries the ^G locus since it has been found in 19 Hb G heterozygotes studied to date. The presence of an -thal-2 heterozygosity and three -chain genes (^0G/) was confirmed among Hb G heterozygotes with lower proportions of this variant. It is likely that the even lower values found in some newborn could arise through defective assembly of ^G- dimers. The presence of an -thal-2 homozygosity and two active -chain genes, one on each chromosome (^0G/⁰), was confirmed among heterozygotes with the higher proportion of Hb G. One of each of these categories was present in each of the three families investigated. This type of variability in the number of active -chain genes due to a heterozygosity or a homozygosity for -thalassemia-2 explains the trimodality of Hb S percentages among heterozygotes and the atypical hematological or biosynthetic features among patients with -thalassemia and sickle-cell syndromes.This research was supported by USPHS Research Grants HLB-05168 and HLB-15158 and by designated research funds of the Veterans Administration. This is Contribution No. 0693 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta.     

Homocysteine stimulates the expression of monocyte chemoattractant protein-1 in endothelial cells leading to enhanced monocyte chemotaxis

The effect of intraperitoneal administration of tocopherol (100 mg/kg wt/24 h) on ascorbate (0.4 mM) induced lipid peroxidation of mitochondria and microsomes isolated from rat liver and testis was studied. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:6 n3 in liver and C20:4 n6 and C22:5 n6 in testis. The lipid peroxidation of liver mitochondria or microsomes produced a significant decrease of C20:4 n6 and C22:6 n3 in the control group, whereas changes in the fatty acid composition of the tocopherol treated group were not observed. The light emission was significantly higher in the control than in the tocopherol treated group. The lipid peroxidation of testis microsomes isolated from the tocopherol group produced a significant decrease of C20:4 n6 , C22:5 n6 and C22:6 n3, these changes were not observed in testis mitochondria. The light emission of both groups was similar. The treatment with tocopherol at the dose and times indicated showed a protector effect on the polyunsaturated fatty acids of liver mitochondria, microsomes and testis mitochondria, whereas those fatty acids situated in testis microsomes were not protected during non enzymatic ascorbateFe²⁺ lipid peroxidation. The protector effect observed by tocopherol treatment in the fatty acid composition of rat testis mitochondria but not in microsomes could be explained if we consider that the sum of C20:4 n6 + C22:5 n6 in testis microsomes is 2-fold than that present in mitochondria.     

Spontaneous mutations changing the raffinose metabolism ofLactobacillus plantarum

Lactobacillus plantarum ATCC 8014 grew poorly on raffinose agar plates, but large mutant colonies appeared in high frequency from a thin film of background growth. The -galactosidase and -galactosidase activities ofL. plantarum ATCC 8014 and a mutant strain were studied in static cultures and pH-controlled fermenter cultures. Both -galactosidase and -galactosidase production were inducible in the parental strain; the induction was not needed in the mutant. The -galactosidase activity of both strains was repressed by glucose but not by -methyl-D-glucoside. The mutant phenomenon might be an obstacle in connection to traditionalLactobacillus identification by means of carbohydrate fermentation.     

Cloning and expression of a member of the Aspergillus niger gene family encoding alpha-galactosidase.

Summary An enzyme with -galactosidase activity and an apparent molecular weight of 82 kDa was purified from culture medium of Aspergillus niger. The N-terminal amino acid sequence of the purified protein shows similarity to the N-terminal amino acid sequence of -galactosidases from several other organisms. Oligonucleotides, based on the N-terminal amino acid sequence, were used as probes to clone the corresponding gene from a EMBL3 gene library of A. niger. The cloned gene (aglA) was shown to be functional by demonstrating that the 82 kDa -galactosidase is absent from a strain with a disruption of the agIA gene, and is over-produced in strains containing multiple copies of the aglA gene. Enzyme activity assays revealed that the 82 kDa -galactosidase A represents a minor extracellular -galactosidase activity in A. niger.     

Proteolytic digestion of α-lactalbumin: Physiological implications

The kinetics of the partial digestion of bovine -lactalbumin (-LA) by trypsin, -chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of -LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)--LA>Zn(II), Ca(II)--LA>apo--LA. The HPLC digestion patterns of Ca(II)--LA and Zn(II), Ca(II)--LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo--LA were quite different, reflecting the existence of the thermally induced denaturation states of apo--LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded -LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of -LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II))-specific site does not cause any drastic changes in the overall structure of -LA. The acidic form of -LA (atpH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or -chymotrypsin at neutralpH. Complexation of -LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to -LA may have physiological significance (e.g., for nutritional transport).On leave from the Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR.     

Establishment of a mutant from human monocytic leukaemia U937 that exhibitsa genetically dominant resistance to TNFα-induced apoptosis

Tumour necrosis factor- (TNF) is a cytokine that induces apoptosis in various cell systems by binding to a TNF receptor (TNFR). To study TNF-induced apoptosis, we isolated and characterized a novel TNF resistant variant, U937/TNF clone II-5, from human monocytic leukaemia U937 cells. The II-5 cells resist apoptosis by TNF and anti-Fas antibody but not by anticancer drugs, such as VP-16 and Ara-C. Somatic cell hybridization between U937 and II-5 showed that the apoptosis resistance to TNF in II-5 was genetically dominant. This dominant mutation in II-5 cells blocks TNF-induced disruption of mitochondrial membrane potential and caspase-3 activation. Expression of TNFR, Fas and Bcl-2 family proteins were not changed in II-5 cells. These results suggest that the apoptosis-resistant II-5 cells could have a functional defect in apoptosis signalling from TNFR to mitochondria and caspase activation. The II-5 cells could be useful in studying the signa lling linkage between TNFR and mitochondria.     

In vivo modification of the cell wall polysaccharide galactomannan of guar transformed with a α-galactosidase gene cloned from senna

Galactomannans are composed of a 1to4 mannan backbone with varying degrees of 1to6 galactose substitutions and are found in the cell walls of legume endosperm. Like other cell wall polysaccharides, many factors controlling the metabolism of galactomannans remain to be elucidated. In the endospermous legume senna (Senna occidentalis) increased -galactosidase activity has previously been observed to coincide temporarily with a decrease of the galactose content of the galactomannan. To evaluate the potential role of -galactosidase for the control of the final galactose content, a -galactosidase gene expressed in immature senna seeds was cloned and transformed into the related high-yielding species guar (Cyamopsis tetragonoloba). The isolated cDNA encoded a 406 amino acid protein with a calculated molecular mass of 44313 Da. The amino acid sequence was 75% identical to the galactomannan hydrolysing -galactosidases from germinating guar and coffee bean. The senna -galactosidase gene was inserted behind a wheat high-molecular-weight glutenin promoter in the vector employed for transformation of guar by Agrobacterium tumefaciens-mediated gene transfer. About 30% of the guar transformants produced endosperm with galactomannans where the galactose content was significantly reduced. After self-fertilization of primary transformants displaying the highest galactose reduction of the galactomannan, endosperms of R₁ plants were analysed demonstrating that this property was inherited stably to the progeny and that it was 100% coupled to the presence of the senna -galactosidase gene. This suggests that -galactosidases can be involved in the determination of the final galactose content of endosperm galactomannans, demonstrating that cell wall polysaccharide biosynthesis can be modified in vivo.