Evidence for a non-antioxidant,dose-dependent role of α -lipoic acid in caspase-3 and ERK2 activation in endothelial cells

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as -lipoic acid and -tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with -lipoic acid and -tocopherol, alone or in combination, prior to incubation with H₂O₂ or staurosporine. -lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H₂O₂ and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with -lipoic acid and/or -tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of Bcl-2, Bax, HSP70 or pERK1/2 or pJNK. -lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with -lipoic acid and -tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of -lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of -lipoic acid as an antioxidant.     

ααααanti-3.7 type II: a new α-globin gene rearrangement suggesting that the α-globin gene duplication could be caused by intrachromosomal recombination

Summary We report here a new human -globin gene rearrangement carrying the two normal, 2 and 1, and two hybrid, 1/2, globin genes in the order 5-2-1/2-1/2-1-3. Both the hybrid genes, subtyped with ApaI and RsaI restriction enzymes, were found to be of the uncommon anti 3.7 type II. The hybrid genes were expressed at the biosynthetic level and their interaction with the -thalassaemia IVS 1 nt 1 GA mutation caused thalassaemia intermedia. We also report a case of an -globin gene rearrangement in the twin of one of the -globin gene carriers; the duplicated gene was of the anti 4.2 type and was associated with the absence of RsaI polymorphism. The singular finding of an -anti 3.7 cluster with two identical rare hybrid genes suggests that the reciprocal unequal recombination causing the -globin gene rearrangements could be of the intra-chromosomal rather than the interchromosomal type.     

Different susceptibilities of complex-, hybrid- and high-mannose-type α1- inhibitor and α1-acid glycoprotein to endo-β-N-acetylglucosaminidase F and peptide:N-glycosidase F

Endo--N-acetylglucosaminidase F (endo F, EC 3.2.1.96) and peptide:N-glycosidase F (PNGase F, EC 3.2.2.18) fromFlavobacterium meningosepticum were used for the deglycosylation of ₁-proteinase inhibitor and ₁-acid glycoprotein carrying oligosaccharide side chains of the complex-, high-mannose- and hybrid-type. High-mannose-and hybrid-type glycoproteins were obtained by the incubation of rat hepatocyte primary cultures with 1-deoxymannojirimycin or swainsonine, respectively. It was found that endo F cleaves hybrid- and high-mannose-type ₁-proteinase inhibitor and ₁-acid glycoprotein at pH 4.5 as well as at pH 8.5 in the presence or absence of 1% octyl--d-glucopyranoside. Complex-type ₁-proteinase inhibitor or ₁-acid glycoprotein were not cleaved by endo F even in the presence of octyl--d-glucopyranoside.PNGase F was found to cleave complex-, hybrid- and high-mannose-type oligosaccharide side chains of ₁-proteinase inhibitor and ₁-acid glycoprotein at pH 4.5 and pH 8.5 in the presence of 0.75% octyl--d-glucopyranoside. The deglycosylation of both protein substrates was very poor without detergents.Abbreviations Endo F endo--N-acetylglucosaminidase F (EC 3.2.1.96) - PNGase F peptide:N-glycosidase F (EC 3.2.2.18) Dedicated to Prof. Dr. Wolfgang Gerok on the occasion of his 60th birthday     

Haemoglobin synthesis in 28 obligatory cases for α-thalassemia traits

Summary In the Far East two types of -thalassemia genes, namely -thalassemia₁ (-thal₁) and -thalassemia₂ (-thal₂) exist. Definite diagnosis of the -thal₁ and -thal₂ traits is very difficult because their hematological findings are minimally abnormal or normal. This study attempts to characterize the heterozygotes by hemoglobin chain synthesis in reticulocytes from obligatory cases of the -thal₁ and -thal₂ traits. Twelve parents of babies with hemoglobin Bart's hydrops fetalis (obligatory -thal₁ trait) had the mean total radioactivity / ratio of 0.76±SD 0.04, while that of 7 normal controls was 1.06±SD 0.04. The / globin chain ratios of 16 cases, who were either parents or offspring of patients with hemoglobin H disease, were found to segregate into 2 groups, i.e. 0.78±SD 0.03 (10 cases) and 0.92±SD 0.03 (6 cases), probably representing the -thal₁ and -thal₂ traits respectively. The hematological data of the first group showed definite hypochromic microcytic red cells, similar to thoseof the parents of the hydrops. The second group had significantly higher mean corpuscular hemoglobin than the first group, compatible with -thal₂ trait. Our globin chain synthesis study thus appears to be capable of discriminating normal, -thal₁ and -thal₂ traits.A preliminary report of the results was presented at the XV Congress of the International Society of Haematology, Israel, September, 1974.     

The effect of culture and membrane potential on Goα expression in neonatal rat cardiac myocytes

The effects of culture and membrane potential on G_o39 expression were examined in neonatal rat cardiac myocytes. During six days of culture, the amount of G_o39 in myocytes increased six-fold. The increase in G_o39 appeared to be programmed, since G_o39 of rat hearts also increased in vivo within three days after birth before declining by six days after birth. Furthermore, the age of the rat from which cardiac myocytes were isolated determined the amount of G_o39 that accumulated in cultured cells with myocytes from two day-old rats producing more G_o39 than myocytes from six day-old rats. In addition, agents which alter membrane potential (KCl and bupivacaine) inhibited the accumulation of G_o39 in cultured myocytes. In an attempt to identify the signaling pathway in which cardiac G_o39 is involved, muscarinic receptor-stimulated inositol phosphate production was examined, but was found to be comparable in myocytes that had six-fold differences in G_o39 content. Thus G_o39 does not appear to couple muscarinic receptors to phospholipase C in rat cardiac myocytes.     

Sialidase of swine influenza A viruses: variation of the recognition specificities for sialyl linkages and for the molecular species of sialic acid with the year of isolation

The sialidase of swine influenza A viruses of N1 and N2 subtypes, isolated from 1930 to 1992, was studied for substrate specificity with ganglio-series, lacto-series type II and GM3 gangliosides containing Neu5Ac2-3Gal, Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. All viral sialidases tested showed that the activity for hydrolysing substrates with Neu5Ac2-3Gal was higher than the activities with Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. When GM1b, GM3 and sialylparagloboside were used as substrates, the earliest strain (A/Wisconsin/15/30 H1N1, isolated in 1930) showed the activity ratio of Neu5Ac2-6Gal to Neu5Ac2-3Gal to be 0.13:0.2, and the ratio Neu5Gc2-3Gal/Neu5Ac2-3Gal to be 0.19:0.37, while those strains isolated from 1978 to 1992 exhibited ratios of 0.29:0.58 for Neu5Ac2-6Gal/Neu5Ac2-3Gal and 0.51:0.76 for Neu5Gc2-3Gal/Neu5Ac2-3Gal. The above results indicate that the substrate specificities of sialidases from swine influenza A viruses towards sialyl linkages and the molecular species of sialic acid are related to the year of isolation, i.e. strains isolated after 1978 exhibited higher activity towards Neu5Ac2-6Gal and Neu5Gc2-3Gal linkages when compared with strains isolated in an earlier year, 1930.Abbreviation Neu5Ac 5-N-acetylneuraminic acid - Neu5Gc 5-N-glycolyneuraminic acid - Gal d-galactose - Glc d-glucose - Cer Ceramide - II³(Neu5Ac)Lac Neu5Ac2-3Gal1-4Glc - GM3(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-4Glc1-Cer - GM3(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-4Glc1-Cer - GM1b(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-3GalNac1-4Gal1-4Glc1-Cer - GMlb(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-3GalNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Ac)nLc4Cer Neu5Ac2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Gc)nLc4Cer Neu5Gc2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV⁶(Neu5Ac)nLc4Cer Neu5Ac2-6Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - TDC taurodeoxycholate.     

High frequency of triplicated α-globin loci and absence or low frequency of α thalassemia in Polynesian Samoans

Summary Most of the population in certain areas of Melanesia have one -globin gene deletion ( thal₂). It is thought that the high frequencies of thal₂ in this population is due to a selective advantage given by malaria infection to carriers of thal₂. We are interested in neighboring Polynesia which, although adjacent to Melanesia, has always been free of malaria due to the absence of the vector anopheles. We studied 60 Polynesian Samoans and 150 Malaysians by restriction endonuclease gene mapping using Eco RI, Bam HI, and Bgl II and hybridization to ³²P-labeled -globin gene probe. Seven among the 60 (11.7%) Samoans had triplicated -globin loci type 1, while none had thal₂. On digestion with Bgl II the third -globin gene was found in an additional 3.7kb fragment in all seven Samoans with triplicated -globin loci, while digestion with Bam HI produced an abnormal elongated 18.2 kb fragment carrying -globin genes in addition to the normal 14.5 kb fragment. None of the Polynesian Samoans had thal₂ or thal₁. Only two of the Malaysians had triplicated -globin loci.     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

Lysine Dendrimers and Their Starburst Polymer Derivatives: Possible Application for DNA Compaction and in vitro Delivery of Genetic Constructs

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)₈-(,-Lys)₄-(,-Lys)₂-(,-Lys)-Ala-NH₂ (D1) and ((Lys)₈-(,-Lys)₄-(,-Lys)₂-,-Lys)-Ala-[Lys(Plm)]₂-Ala-NH₂ (D2), as well as the starburst polymeric derivatives of D1, (pVIm) ₈ -D1 and (pLys) _n -D1, containing poly(N-vinylimidazole) and polylysine chains single-point bound to the dendrimer amino groups. The conditions of dendrimer–plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on mouse C2C12 myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex.     

Heterogeneity of the α-globin gene defects in German α-thalassemia affected families

Summary Analysis of -thalassemia syndromes in several German families revealed DNA deletion as well as nondeletion forms as the molecular basis for the defects. Thus, the -thalassemia haplotype was identified as the (–)^3.7 rightward deletion form, and the region of the putative recombination process generating such a deletion was further characterized. In addition three different ° haplotypes, (--)^MED, (--)^>26, and ()^T, could be detected using -and -globin gene-specific probes.     

Expression of receptor protein tyrosine phosphatase agr; mRNA in human prostate cancer cell lines

Receptor protein tyrosine phosphatase (RPTP) is transmembrane protein phosphatases, and has been proposed to be involved in the differentiation of the neuronal system. In the present study, we demonstrated the expression of RPTP mRNA in several normal human tissues. We further investigated the regulation of expression of RPTP mRNA in epithelial cells utilizing three commercially available human prostate cancer cell lines LNCaP, PC-3 and DU145. This is because these cells exhibit different levels of differentiation, defined by the expression of a tissue-specific differentiation antigen, prostatic acid phosphatase (PAcP), and their androgen sensitivity. LNCaP cells express PAcP and are androgen-sensitive cells, while PC-3 and DU145 cells do not express PAcP and are androgen-insensitive cells. Northern blot analyses revealed that, in LNCaP cells, fetal bovine serum (FBS) and 5-dihydrotestosterone (DHT) down-regulates RPTP mRNA expression, similar to the effect on PAcP. Contrarily, FBS up-regulated the RPTP mRNA level in PC-3 and DU145 cells. In LNCaP cells, sodium butyrate inhibited cell growth and up-regulated RPTP as well as PAcP mRNA expression. Although, sodium butyrate also inhibited the growth of PC-3 and DU145 cells, the level of RPTP mRNA was decreased in PC-3, while increased in DU145 cells. Thus, data taken together indicate that the expression of RPTP is apparently regulated by a similar mechanism to that of PAcP in LNCaP cells.     

N-Carbamoyl-α-Amino Acids Rather than Free α-Amino Acids Formation in the Primitive Hydrosphere: A Novel Proposal for the Emergence of Prebiotic Peptides

Our previous kinetic and thermodynamic studies upon the reactional system HCHO/HCN/ NH₃ in aqueous solutions are completed. In the assumed prebiotic conditions of the primitive earth ([HCHO] and [HCN] near 1 g L^–1, T = 25 °C, pH = 8, [NH₃] very low), this system leads to 99.9% of -hydroxyacetonitrile and 0.1% of -aminoacetonitrile (precursor of the -amino acid). The classical base-catalyzed hydration of nitriles, slow and not selective, can not modify significantly this proportion. On the contrary, we found two specific and efficient reactions of -aminonitriles which shift the initial equilibrium in favor of the -aminonitrile pathway. The first reaction catalyzed by formaldehyde generates -aminoamides, precursors of -aminoacids. The second reaction catalyzed by carbon dioxide affords hydantoins, precursors of N-carbamoyl--aminoacids. In the primitive hydrosphere, where the concentration in carbon dioxide was estimated to be higher than that of formaldehyde, the formation of hydantoins was consequently more efficient. The rates of hydrolysis of the -aminoacetamide and of the hydantoin at pH 8 being very similar, the synthesis of the N-carbamoyl--amino acid seems then to be the fatal issue of the HCHO/HCN/NH₃ system that nature used to perform its evolution. These N-protected -amino acids offer new perspectives in prebiotic chemistry, in particular for the emergence of peptides on the prebiotic earth.     

The use of 1JCαHα coupling constants as a probe for protein backbone conformation

Summary Simple pseudo-3D modifications to the constant-time HSQC and HCACO experiments are described that allow accurate (±0.5 Hz) measurement of one bond J_CH coupling constants in proteins that are uniformly enriched with ¹³C. An empirical ,-surface is calculated which describes the deviation of ¹J_CH from its random coil value, using 203 ¹J_CH values measured for residues in the proteins calmodulin, staphylococcal nuclease, and basic pancreatic trypsin inhibitor, for which and are know with good precision from previous X-ray crystallographic studies. Residues in -helical conformation exhibit positive deviations of 4–5 Hz, whereas deviations in -sheet are small and, on average, slightly negative. Data indicate that ¹J_CH depends primarily on , and that ¹J_CH may be useful as a qualitative probe for secondary structure. Comparison of ¹J_CH coupling constants measured in free calmodulin and in its complex with a 26-aminoacid peptide fragment of myosin light-chain kinase confirm that the calmodulin secondary structure is retained upon complexation but that disruption of the middle part of the central helix is even more extensive than in free calmodulin. Supplementary material available from the authors: One table listing 352 ¹J_CH and ¹J-values, together with ,-values for 203 residues of known conformation. Two figures showing (a) a Ramachandran plot of the ,-values of 203 residues used in deriving ¹J(,), and (b) the r.m.s.d. ¹J(,) distribution.     

Pseudo vitamin B12 or 5-hydroxybenzimidazolyl-cobamide are the corrinoids found in methanogenic bacteria

Thirteen species of methanogenic bacteria were analyzed for corrinoids. Pseudo vitamin B₁₂ (Co-[-(7-adenyl)]-cobamide) was the predominant cobamide of methanococcales and Methanoplanus. All other methanogens contained factor III (Co-[-(5-hydroxybenzimidazolyl)]-cobamide). Vitamin B₁₂ (Co-[-(5,6-dimethylbenzimidazolyl)]-cobamide) was not detected in any of these archaebacteria. Their cobamide content was 100 to 1400 nmol per gram cell dry weight, indicating that abundant cobamides are essential for methanogens.     

Synthesis of N-linked pentasaccharides with isomeric glycosidic linkage

As part of a program to explore the structural requirement of N-glycans in the carbohydrate-mediated biological interactions, N-linked pentasaccharide core structure was stereochemically modified in terms of glycosidic linkage. Three isomers, -D-Man-(13)-[-D-Man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, -D-Man-(13)-[-D-Man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, and -D-Man-(13)-[-D-man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, were synthesized. Synthesis of the pentasaccharide with natural linkage is also described.     

Detection of a new hybrid α2 globin gene among American Blacks

Summary We have examined the globin gene complex for 49 individuals with -thalassemia-2 (–^3.7). Crossovers resulting in -thalassemia-2 (type I) were observed in all 57 chromosomes with the –^3.7 defect. Except for one -thalassemia-2 chromosome, all were linked to the absence of an Rsa I restriction site located 0.7 kb 5 to the 2-globin gene; this polymorphic site was observed for 10 of 38 non--thalassemia chromosomes from Black Americans. In four Black families with a heterozygous -thalassemia-2 [–^3.7 (I)], an Apa I restriction site has been identified in the IVS-2 of the 2 gene of the normal chromosome (labeled the ^*2 gene). The ^*2 gene of one Black subject was cloned and a segment located 5 to the Cap site as well as the IVS-2, exon 3, and a 3 segment were sequenced. The data show that the ^*2 gene is an 2 gene except for a segment between nucleotides (nts) 580–81 and nt 509 (Cap site=nt 1), and perhaps as far upstream as nt-634, which has an 1 sequence. This ^*2 hybrid gene probably originated through a double crossover; the structural identity of its IVS-2 with that of the 1 gene adequately explains the presence of the Apa I restriction site.     

The minimal polymorphism of class II Eα chains is not due to the functional neutrality of mutations

Given the extensive allelic amino acid sequence polymorphism present in the first domain of A, A, and E chains and its profound effects on class II function, the minimal polymorphism in the mouse E chain (and in its human homologue DR) is paradox. Two possible explanations for the lack of polymorphism in E are: (1) the E chain plays such a uniquely critical structural/functional role in antigen presentation, T-cell activation, repertoire selection, and/or pairing with E or other proteins for expression that it cannot vary, and mutations are selected against; (2) the E chain plays a less significant role than the outer domains of other major histocompatibility complex (MHC) proteins in determining the interactions with processed peptides or with T-cell receptor (TCR), so there is no selective pressure to maintain new mutations. To explore this question we compared the ability of transfectants expressing wild type (wt) EE ^d and mutant E wt E ^d proteins to present peptides and bacterial superantigens to T-cell hybridomas. Mutations at the E amino acid positions 31, 52, and 65&66, to residues that represent allelic alternatives in A chains, significantly reduced activation of peptide-specific T hybridomas, and mutations at 71 sometimes enhanced T-cell stimulation. None of the E mutations reduced, and some enhanced, superantigen stimulation of T-cell hybridomas. These results argue against the hypothesis that E chains are minimally polymorphic because mutations in E are functionally neutral.     

An alpha-L-fucosidase from Thermus sp. with unusually broad specificity

An -L-fucosidase (E.C. 3.2.1.51) exhibiting a wide aglycon specificity expressed in ability of cleaving 1 6-, 1 3-, 1 4-, and 1 2-O-fucosyl bonds in fucosylated oligosaccharides, has been isolated from culture filtrate of Thermus sp. strain Y5. The -L-fucosidase hydrolyzes p-nitrophenyl -L-fucopyranoside with V _max of 12.0 ± 0.1 M/min/mg and K _m = 0.20 ± 0.05 mM and is able to cleave off about 90% of total L-fucose from pronase-treated fractions of fucosyl-containing glycoproteins and about 30% from the native glycoproteins. The purified enzyme is a tetramer with a molecular mass of 240 ± 10 kDa consisting of four identical subunits with a molecular mass of 61.0 ± 0.5 kDa. The N-terminal sequence showed homology to some -L-fucosidases from microbial and plant sources. Hydrolysis of p-nitrophenyl -L-fucopyranoside occurs with retention of the anomeric configuration. Transglycosylating activity of the -L-fucosidase was demonstrated in reactions with such acceptors as alcohols, N-acetylglucosamine and N-acetylgalactosamine while no transglycosylation products were observed in the reaction with p-nitrophenyl -L-fucopyranoside. The enzyme can be classified in glycosyl hydrolase family 29.     

Large-scale expression of recombinant sialyltransferases and comparison of their kinetic properties with native enzymes

Values ofK _m were determined for three purified sialyltransferases and the corresponding recombinant enzymes. The enzymes were Gal1-4GlcNAc 2-6sialyltransferase and Gal1-3(4)GlcNAc 2-3sialyltransferase from rat liver; these enzymes are responsible for the attachment of sialic acid to N-linked oligosaccharide chains; and the Gal1-3GalNAc 2-3sialyltransferase from porcine submaxillary gland that is responsible for the attachment of sialic acid to O-linked glycoproteins and glycolipids. A procedure for the large scale expression of active sialyltransferases from recombinant baculovirus-infected insect cells is described. For the liver enzymes values ofK _m were determined using rat and human asialo₁ acid glycoprotein andN-acetyllactosamine as variable substrates; lacto-N-tetraose was also used with the Gal1-3(4)GlcNAc 2-3sialyltransferase. Antifreeze glycorprotein was used as the macromolecular acceptor for the porcine enzyme. Values forK _m were also determined using CMP-NeuAc as the variable substrate.Abbreviations NeuAc N-acetylneuraminic acid - Gal galactose - GlcNAc N-acetylglucosamine Enzymes: Gal1-4GlcNAc 2-6sialyltransferase, EC 2.4.99.1; Gal1-3(4)GlcNAc 2-3sialyltransferase, EC 2.4.99.5; Gal1-3GalNAc 2-3sialyltransferase, EC 2.4.99.4.