Are polyphosphoinositides associated with glycophorin in human erythrocyte membranes?

Glycophorin prepared by a lithium di-iodosalicylate-extraction/phenol-partition method was rich in polyphosphoinositides (phosphatidyl-myo-inositol 4-phosphate and phosphatidyl-myo-inositol 4,5-bisphosphate), but glycophorin extracted by Triton X-100 showed no such enrichment. The enrichment observed in the former preparations appeared not to be caused by pre-existing association between glycophorin and polyphosphoinositides in the human erythrocyte membrane, but to be largely a consequence of the preparative procedures.     

The molecular basis of β-thalassemia in Turkey

Summary By using oligonucleotide hybridization, restriction endonuclease analysis and direct sequencing of amplified genomic DNA, we have been able to characterize 18 different mutations in the -globin genes of 161 thalassemia homozygotes and 107 -thalassemia heterozygotes from Turkey (429 -thalassemia chromosomes). Previous studies dealing with -thalassemia in Mediterranean countries have shown that, in most Mediterranean populations, only a few mutations are prevalent. In contrast, -thalassemia in Turkey does not seem to be associated with a few predominant mutations. The six most frequent alleles, IVS-I-110 (GA), IVS-I-6(TC), FSC-8 (-AA), IVS-I-1(GA), -30(TA) and FSC-5 (CT), account for only 69.3% of the disease genes; indeed, all 26 mutations assayed represent 85.8% of the disease genes, confirming the considerable molecular heterogeneity of -thalassemia among Turks, and indicating the possible presence of rare, previously undefined, mutations in the population. Two mutations observed in this study, IVS-I-116 (TG) and Cd44(-C), have not been reported in the Turkish population to date. Since preventive medical services, such as genetic counseling and prenatal diagnosis, are greatly improved by detailed knowledge of the molecular pathology of thalassemia, we strongly believe that the presented data will facilitate the intended establishment of a prenatal diagnosis center, based on DNA analysis, in Turkey.     

Blood antioxidant status and urinary levels of catecholamine metabolites in β-thalassemia

It has been reported that iron overload in β-thalassemia leads to an enhanced generation of reactive oxygen species and to oxidative stress. We have studied the oxidant/antioxidant imbalance in the blood of 48 transfusion-dependent β-thalassemic patients (TLP) (17 males, 31 females, 11–22 year), under chelation therapy, and in 40 sex and age matched healthy controls (CTR). Plasma and lymphocyte levels of vitamin E (Vit E), ubiquinol (CoQ₁₀H₂), ubiquinone (CoQ₁₀), plasma concentrations of vitamin A (Vit A), β-carotene, lycopene, vitamin C (Vit C), total thiols, fatty acid patterns of phospholipids (PL-FA), and plasma and urinary markers of lipoperoxidation (TBA-RM, conjugated dienes, and azelaic acid (AZA), as well as the urinary levels of catecholamine and serotonin metabolites, were evaluated by gas chromatography-mass spectrometry (GC-MS), HPLC and spectrophotometry. Routine laboratory blood analyses were performed on the same samples; 39/48 TLP were HCV positive. Blood samples were collected just before transfusion, the 24 h urine samples the day before. Our results clearly showed that a severe oxidative stress occurs in the plasma of TLP in comparison with CTR. In fact, the levels of lipophilic antioxidants and ascorbate were severely depleted: CoQ₁₀H₂ (-62.5%), total CoQ₁₀ (-35.1%), Vit E (-43.8%, β-carotene (-31.1%), lycopene (-63.7%), Vit A (-35.9%), Vit C (-23.1%). The impairment of the antioxidant status was associated with elevated plasma levels of by-products of lipoperoxidation and urinary concentrations of catecholamine metabolites and of AZA, indicating a high degree of both neurological stress and lipoperoxidation. A significant positive correlation was found between vitamin E and non-transferrin-bound iron (NTBI) (r = -0.81; p < 0.001), while no correlation was found between antioxidant depletion and ferritin serum levels, average blood consumption, or the presence of clinical complications. The administration of selective antioxidants along with an appropriate diet might represent a promising way of counteracting oxidative damage and its deleterious effects on the progression of the disease.     

The spectrum of β-thalassemia mutations in northern and northeastern Thailand

Summary A total of 123 -thalassemia genes from northern (n = 113) and northeastern (n = 10) Thailand were examined. Using five oligonucleotide probes, the mutation in 108 genes (88%) was identified: 50 nonsense 17, 49 frameshift 41-42, 4-28(AG), 2IVS1 nt5(GC), 2IVS2 nt654, and 1 deletion removing the entire -globin gene. The nonsense 17 mutation (n = 39) was linked to a single haplotype, whereas the frameshift 41-42 mutation occurred with several haplotypes. The results of the present study indicate that prenatal diagnosis of clinically important -thalassemia syndromes using a limited set of oligonucleotides is feasible in approximately 80% of affected families in northern Thailand and most of the families with -thalassemia-Hb E disease in northeastern Thailand.     

Molecular characterization of β-thalassemia in Azerbaijan

We have analyzed the -thalassemia mutations in 99 chromosomes of 49 adults with -thalassemia major and of one with Hb S--thalassemia, who are regular patients at a large hematology clinic in Bakü, Azerbaijan. A total of 20 different mutants were identified; three [frameshift at codon 8 (-AA); IVS-II-I (GA); IVS-I-110 (GA)] were present in about two-thirds of all chromosomes. Most alleles are the same as found in Mediterranean populations; a few have an Asian origin or come from Kurdistan, Lebanon, Saudi Arabia, or a black population. One mutant [frameshift at codons 82/ 83 (-G)] might be specific for the Azerbaijanian population. Nearly all patients were transfused, which made quantitation of Hb F impossible; high^G values were present in the Hb F of those patients whose -thalassemia chromosome carried the C T mutation at position — 158 in the promoter of the ^G-globin gene.     

Molecular characterization of β-thalassemia in Czechoslovakia

Summary We have identified different -thalassemia mutations in 93 members of 34 families of Czech or Slovakian descent using gene amplification, hybridization with specific ³²P-labeled oligonucleotide probes, sequencing of amplified DNA, and gene mapping. The GA mutation at IVS-I-1 was found in 18 families; other Mediterranean mutations were IVS-II-1 (GA), IVS-II-745 (CG), IVS-I-110 (GA), and codon 39 (CT); these were present in 9 additional families. The GT mutation at codon 121, known to cause Heinzbody -thalassemia, was present in 3 families, and the frameshift at codons 82/83 (-G), first described in the Azerbaijanian population, in 2 families. A newly discovered allele was a frameshift at codons 38/39 (-C). One -thalassemia allele was incompletely characterized. We observed in 2 families a TC mutation at position +96 UTR (untranslated region) relative to the termination codon; this mutation likely is a rare polymorphism, -Thalassemia was rare; only one person carried the -^3.7 heterozygosity, and one other had a yet to be identified -thalassemia-1, while seven had the ^{anti 3.7} triplication.     

Molecular characterization of β-thalassemia in Hungary

We have identified seven different -thalassemia mutations and one -thalassemia determinant (the Sicilian type) in 32 members of 17 Hungarian families. The most common mutation is the IVS-I-1 (GA) change; its high frequency is comparable to that observed in neighboring Czechoslovakia. Additional mutations are of Mediterranean origin. One rare mutation (initiation codonATGGTG) was identified as an independent mutation because of the absence of known polymorphisms in the -globin gene. One new frameshift at codon 51 (-C) was observed in a single individual; hematological data were as expected for a °-thalassemia heterozygosity.     

The non-protein amino acid β-N-methylamino-l-alanine in Portuguese cyanobacterial isolates

The tailor made amino acid β-N-methyl-amino-L-alanine (BMAA) is a neurotoxin produced by cyanobacteria. It has been associated with certain forms of progressive neurodegenerative disease, including sporadic Amyotrophic Lateral Sclerosis and Alzheimer's disease. Some different reports of BMAA in cyanobacterial blooms from lakes, reservoirs, and other water resources have been made by different investigators. We here report the detection of BMAA of both free and protein-bound produced by cyanobacteria, belonging to the Chroococcales, Oscillatoriales and Nostocales ordered. We use a rapid and sensitive HPLC-FD method that utilizes methanol elution and the Waters AQC Tag chemistry. On other hand, we have used three different assay procedures for BMAA extraction from cyanobacteria: Trichloroacetic acid (TCA), Methanol/Acetone and hydrochloric acid (HCl). All assays let successfully detect BMAA in all cyanobacteria samples analyzed. Nevertheless, with TCA and HCl extraction procedures the highest BMAA values, for free as well as protein-bound BMAA were detected. BMAA content could not be related to the taxonomy of the isolates or to their geographical origin, and no correlation between free and protein-bound BMAA concentrations were observed within or between taxonomic groups. These data offer confirmation of the taxonomic and geographic ubiquity of BMAA from naturally occurring populations of cyanobacteria, for the first time reported for estuaries.     

The effect of α2,6-linked sialic acid on anti-IgM antibody-induced apoptosis in Ramos cells

Apoptosis in B cells is induced through the B cell antigen receptor (BCR) and affects the sialic acid recognition molecules on B cells. We investigated the effects of ₁-acid glycoprotein (AGP), which mainly contains 2,6-linked sialic acid, on anti-IgM antibody (Ab)-induced apoptosis in Ramos cells, which are derived from Burkitt's lymphoma. When Ramos cells were incubated with anti-IgM-Ab in plates coated with AGP, neuraminidase-digested AGP (asAGP) or 2,3-sialylated AGP (2,3AGP), apoptosis was suppressed only in those coated with AGP. We also studied the effects of CD22, which is expressed on the surface of mature B cells and binds to sugar chains containing 2,6-linked sialic acid, with anti-CD22 monoclonal antibody (mAb). Anti-CD22mAb enhanced anti-IgM Ab-induced apoptosis in Ramos cells. These contradictory results suggested that the recognition molecules for 2,6-linked sialic acid on AGP, which inhibits B-cell apoptosis, is distinct from CD22, or that different binding domains of CD22 between 2,6-linked sialic acid and anti-CD22 mAb exert opposite functions of suppression or enhancement to anti-IgM Ab-induced B cells.     

Bone disease in children with homozygous β-thalassemia

The histological features of thalassemic bone are imperfectly known, and the roles of bone marrow hyperactivity, iron overload or vitamin D deficiency in the pathogenesis of the disease are not clearly identified. In this study we examined iliac crest biopsies from 17 transfusion-dependent children with homozygous β-thalassemia and severe radiological skeletal thalassemic changes, including widening of medullary spaces and osteoporosis. Rachitic lesions were not observed. Serum ferritin concentrations were increased in all but one subject. Iron deposits were histochemically detected in bone marrow, at the marrow-bone interface, along cement lines and mineralizing perimeters. Minor changes were present in trabecular bone, and osteomalacia was absent. By contrast, cortical bone exhibited severe changes including fissures and focal mineralization defects. Plasma 25-hydroxyvitamin D (25(OH)D) concentrations measured during the winter (December–May, 6.5 ± 4.9 ng/ml, mean ± SD, n = 6) and during the summer (June–November, 13.8 ± 8.4 ng/ml, n = 9) did not differ from those of age-matched children living in the same country. Seven patients had moderate hypocalcemia but no biological signs suggestive of vitamin D deficiency: all had normal alkaline phosphatase activity, normal or slightly elevated plasma phosphate, only two had low plasma 25(OH)D concentrations and two others supranormal values of plasma immunoreactive parathyroid hormone.These results show that iron overload and vitamin D deficiency do not seem to play an important role in the pathogenesis of thalassemic bone disease, which is characterized by cortical lesions probably related to marrow hyperactivity.     

Molecular characterization of β-thalassemia mutations in Egypt

Summary The relative frequency of different -thalassemia mutations and their association with -globin haplotypes were studied in patients from the Nile delta region, Egypt, by means of the polymerase chain reaction, oligonucleotide hybridization and restriction analysis. We found that 8 mutations account for 77% of -thalassemia chromosomes in this population, the commonest being IVS-1 nt 110, IVS-1 nt 6 and IVS-1 nt 1. Each mutation was associated with a specific haplotype, with the exception of IVS-1 nt 110, found on 3 different chromosomal backgrounds. Our data show that testing for the 8 detectable mutations makes feasible prenatal diagnosis in 65% of at risk couples and exclusion testing in an additional 25% of cases.     

The mechanism of the periodate–thiobarbituric acid reaction of sialic acids

1. The chromogen formation from N-acetylneuraminic acid in the periodate-thiobarbituric acid reaction was investigated. Measurement of periodate consumption showed an uptake of approx. 3moles/mole of substrate in neutral as well as in strongly acidic solution. Therefore the chromogen beta-formylpyruvic acid is not a direct product of the periodate oxidation; it is presumed to be formed from the true oxidation product, a hexos-5-uluronic acid, by aldol splitting during the reaction in hot acidic solution with thiobarbituric acid. 2. Methyl (methyl beta-l-threo-hexos-4-enepyranosid)uronate, an analogue of the pre-chromogen, has been shown to yield with thiobarbituric acid in acidic solution a pigment exhibiting an identical absorption spectrum and showing the same behaviour on paper chromatography as the pigment obtained from N-acetylneuraminic acid in the periodate-thiobarbituric acid assay. 3. The substitution at C-2 of methoxyneuraminic acid does not inhibit the periodate-thiobarbituric acid reaction. In neutral solution methoxyneuraminic acid is oxidized by periodate to a substance that reacts readily with thiobarbituric acid in acidic solution. When periodate oxidation is attempted in acidic solution, protonation of the amino group protects this group against oxidation, rendering methoxyneuraminic acid negative in the assay systems of Warren (1959a,b) and Aminoff (1959, 1961).     

Serum total sialic acid levels in smokers and users of smokeless tobacco in form of oral powder (Maraş powder)

Summary Smokeless tobacco (ST) is widely used as chewing tobacco and as oral snuff in the world. Also, in Kahramanmaraş, a city in Southern Turkey, ST used as ‘oral powder’ or ‘Maraş Powder’ is consumed widely instead of cigarette smoking. The aim of this study was to search the effect of ST use on serum total sialic acid (TSA) and to compare the serum TSA levels in smokers and ST users. The study was performed at the Department of Chemistry-Biochemistry, University of Kahramamaraş Sutcu Imam, Turkey, in 2003. Serum samples obtained from smokers (Group I), Maraş powder users (Group II), and healthy control subjects (Group III) who were nonsmokers and nonusers ST. Individuals who were smokers and ST users were classified into subgroups with respect to amount of consumed cigarette or oral powder. Serum TSA was measured with the Denny’s colorimetric method. The TSA concentrations were significantly higher in the sera of smokers (p < 0.001) and Maraş powder users (p < 0.001) than those of control subjects. The mean serum TSA level was found to be lowest in the control group and highest in the Maraş Powder users. But, there was no significant difference in serum TSA levels between smokers and Maraş powder users (p > 0.05). We can conclude from the results obtained that serum TSA was affected by ST use as seen in smokers. This finding may be an indication of harmful effects of ST use as Maraş powder as well as cigarette smoking.     

Histochemical methods for acid β-galactosidase: Technics for semipermeable membranes

Summary Indigogenic and azocoupling reactions for the detection of acid -galctosidase in unfixed cold microtome sections adherent to semipermeable membranes are described. The indigogenic method is the method of choice. The described procedure prevents the leakage of the enzyme activity of sections (the diffusion is limited to the closest surroundings of the actual localization of enzyme activity) and is recommended as a routine method in studies concerning acid -galactosidase.     

The acid and enzymic hydrolysis of O-acetylated sialic acid residues from rabbit Tamm–Horsfall glycoprotein

Rabbit Tamm-Horsfall glycoprotein and bovine submaxillary glycoprotein were both found to contain sialic acid residues which are released at a slow rate by the standard conditions of acid hydrolysis. These residues are also resistant to neuraminidases from Vibrio cholerae and Clostridium perfringens. This behaviour was attributed to the presence of O-acetylated sialic acid, since the removal of O-acetyl groups by mild alkaline treatment normalized the subsequent release of sialic acid from rabbit Tamm-Horsfall glycoprotein by acid and by enzymic hydrolysis. Determination of the O-acetyl residues in rabbit Tamm-Horsfall glycoprotein indicated that on average two hydroxyl groups of sialic acid are O-acetylated, and these were located on the polyhydroxy side-chain of sialic acid or on C-4 and C-8. These findings confirm the assumption that certain O-acetylated forms of sialic acid are not substrates for bacterial neuraminidases. Several explanations have been suggested to explain the effect of O-acetylation of the side-chain on the rate of acidcatalysed hydrolysis of sialic acid residues.     

“Just a spoonful of sugar...”: import of sialic acid across bacterial cell membranes

Eukaryotic cell surfaces are decorated with a complex array of glycoconjugates that are usually capped with sialic acids, a large family of over 50 structurally distinct nine-carbon amino sugars, the most common member of which is N-acetylneuraminic acid. Once made available through the action of neuraminidases, bacterial pathogens and commensals utilise host-derived sialic acid by degrading it for energy or repurposing the sialic acid onto their own cell surface to camouflage the bacterium from the immune system. A functional sialic acid transporter has been shown to be essential for the uptake of sialic acid in a range of human bacterial pathogens and important for host colonisation and persistence. Here, we review the state-of-play in the field with respect to the molecular mechanisms by which these bio-nanomachines transport sialic acids across bacterial cell membranes.     

Characterization of subfractions of β2-glycoprotein I: Evidence for sialic acid microheterogeneity

• 1.1. β₂-Glycoprotein I is a sialic acid microheterogeneous protein and contains on the average 11 mol sialic acid/mol.
• 2.2. Linear correlation was found between sialic acid content and pI of isolated subfractions.
• 3.3. Asialo-β₂2-glycoprotein I consists of 2 isoforms. Each of which can originate from the same subfraction.
• 4.4. The isolated subfractions exhibited almost the same amino acid composition.

     

Fetal hemoglobin in normal adults and β-thalassemia heterozygotes

Summary A recently developed high performance liquid chromatographic (HPLC) procedure using a weak cation exchanger (PolyCAT) in columns of different sizes was used to quantify fetal hemoglobin (Hb F) in blood of normal adults and -thalassemia (-thal) heterozygotes with ten different types of mutations. Preparative PolyCATHPLC greatly facilitated the characterization of isolated Hb F, i.e., the determination of the relative quantities of the ^G and ^A chains. The method is accurate and allows quantitation of Hb F at the 0.5% level; preparative PolyCAT-HPLC allows isolation of (nearly) pure Hb F from blood samples with low (< 1%) Hb F. Adult Hb F levels were determined in 69 normal adults (including 24 diabetics); Hb F levels fell below 1% except for subjects with abnormal -^G-^G arrangement and a CT mutation at position -158 relative to the Cap site of both ^G genes. The effect of the same mutation in the normal-^G-^A-arrangement was variable. Certain -thal mutations (namely, those at positions -29;-88; IVS-I-1; IVS-II-1) were associated with high Hb F levels in heterozygotes, while those at nucleotide (nt) positions IVS-I-6; IVS-I-110; codon 24; codon 39; codons 41/42; IVS-II-745 were not. ^G values varied and often fell into two groups (high ^G and low ^G); high ^G values were not associated with high Hb F values. The chromatographic procedure is ideally suited for Hb A₂ quantitation. Average values of Hb A₂ in -thal heterozygotes with any one of nine of the ten mutations were twice that of normals; the one exception was the -thal heterozygote with the IVS-I-6 (TC) mutation with an average low Hb A₂ value of 3.6%.     

Mediterranean types of β-thalassemia in the German population

Summary Forty -thalassemia genes from unrelated German heterozygotes with no known foreign ancestry were examined using the oligonucleotide technique and DNA restriction analysis, with the aim of determining the contribution of Mediterranean -thalassemia mutations to the prevalence of this trait in the German population. Of the 40 -thalassemia genes, 26 were identified as Mediterranean types (20 39 nonsense, 3 IVS2 nt 110, 2 IVS2 nt1, 1 IVS1 ntl GA). The geographic distribution of the birthplaces of the probands' grandparents revealed no difference in the proportion of Mediterranean and unidentified -thalassemia genes in the west and the north of Germany.