Mutation analysis of glucose-6-phosphate dehydrogenase (G6PD) variants in Costa Rica

Summary Glucose-6-phosphate dehydrogenase (G6PD) deficiency has previously been reported among both the black and white populations of Costa Rica. All 28 G6PD A — samples were found to be of the common G6PD A-^376G/202Atype. A previously described mutation associated with nonspherocytic hemolytic anemia, G6PD Puerto Limón, was found to be due to a GA transition at nucleotide (nt) 1192, causing a glulys substitution. Mutations in this region of the G6PD molecule seem invariably to be associated with chronic hemolytic anemia. G6PD Santamaria had been described previously in two unrelated white subjects. We found that both did, indeed, have the same mutations. In this variant the AG substitution at nt 376 that is characteristic of G6PD A was present, but an AT mutation at nt 542, apparently superimposed on the ancient G6PD A mutation, resulted in an aspval substitution. Thus, the gain of a negative charge at amino acid 126 was counterbalanced by the loss of a charge at amino acid 181, giving rise to a variant with the G6PD A mutation but with normal electrophoretic mobility.     

Occurrence of reducing terminal N-acetylglucosamine 3-sulfate and fucosylated outer chains in acidic N-glycans of porcine zona pellucida glycoproteins

Structures of acidic N-glycans released from porcine zona pellucida glycoproteins by hydrazinolysis were studied. The results indicated that the acidic glycans are of mono- to tetraantennary complex-type with and without N-acetyllactosamine repeating units. Sulfated residues are not only located at the C-6 position of GlcNAc included in the N-acetyllactosamine repeating units, but also at the C-6 position of GlcNAc in the non-repeated antennae and at the C-3 position of reducing terminal GlcNAc residue. Analysis of the oligosaccharide fragments released by endo--galactosidase digestion and by hydrazine/nitrous acid treatment also revealed that various sulfated and non-sulfated forms of fucosylated structures such as Fuc12Gal14(±SO–36)GlcNAc (type 2H), Gal14(Fuc13)(±SO–36)GlcNAc(Lex) and Fuc13 or 4(±SO–36)GlcNAc, are expressed in the repeated outer chain moieties.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Structures of the N-linked sugar chains in the PAS-6 glycoprotein from the bovine milk fat globule membrane

The structures of the N-linked sugar chains in the PAS-6 glycoprotein (PAS-6) from the bovine milk fat globule membrane were determined. The sugar chains were liberated from PAS-6 by hydrazinolysis, and the pyridylaminated sugar chains were separated into a neutral (6N) and two acidic chains (6M and 6D), the acidic sugar chains then being converted to neutral sugar chains (6MN and 6DN). 6N was separated into two neutral fractions (6N13 and 6N5.5), while 6MN and 6DN each gave a single fraction (6MN13 and 6DN13). The structure of 6N5.5, which was the major sugar chain in PAS-6, is proposed to be Man16 (Man13) Man14GlcNAc14GlcNAc-PA; 6N13, 6MN13 and 6DN13 are proposed to be Gal13Gal14GlcNAc12Man16 (Gal13Gal14GlcNAc12Man13) Man14GlcNAc14 (Fuc16)GlcNAc-PA;6M and 6D had 1 or 2 additional NeuAc residues at the non-reducing ends of 6MN13 and 6DN13, respectively. © 1998 Rapid Science Ltd     

A model of associative memory in the brain

A model of associative memory for time varying spatial patterns is proposed and simulated on a digital computer. This is a network composed of many neuron-like elements, and shows an ability for associative memory similar to that of the brain.Suppose a number of sequences of spatial patterns are presented to this network, for example, 12345, ABC, and so on. Then, these patterns are memorized in the network. After that, if any part of one of these sequences, say 23, is presented to the circuit, the rest of the sequence, 45, is recalled following to it. It resembles to such a situation — if we hear a part of a melody which we have memorized in the past, the rest of the melody is recalled even after it is stopped half-way. Although the recalled patterns are not always 100% correct, they are not completely destroyed even if the presented patterns are imperfect.     

α-Galactosidase gene mutations in Fabry disease: heterogeneous expressions of mutant enzyme proteins

Five point mutations were identified in unrelated Japanese Fabry disease hemizygotes: three new missense mutations, C142Y (⁴²⁵ G A), A156V (⁴⁶⁷ C T), and L166V (⁴⁹⁶ C G) in exon 3; one new splice site mutation at the 3 end of the consensus sequence in exon 4; one previously reported nonsense mutation, W44X (¹³¹ G A). C142Y expressed 50% of the normal enzyme protein in COS-1 cells, but catalytic activity was not detected. Both A156V and L166V expressed significant amounts of residual enzyme activity (6.7% and 9.8%) and enzyme proteins (10% each), the latter were more thermolabile at neutral pH than at acid pH, in vitro.     

Effects of supplied cinnamic acids and biosynthetic intermediates on the anthocyanins accumulated by wild carrot suspension cultures

Anthocyanins isolated and characterized from the wild carrot suspension cultures used here were 3-O--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D<-galactopyranosylcyanidin (1), 3-O-[-D- xylopyranosyl-(12)--D-galactopyranosyl]cyanidin (2), 3-O-(6-O-sinapoyl)--D-glucopyranosyl-(16)-[-D- xylopyranosyl-(12)-]-D-galactopyranos ylcyanidin (3), 3-O-(6-O-feruoyl)--D-glucopyranosyl-(16)-[- D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (4), 3-O-(6-O-coumaroyl)--D-glucopyranosyl-(16)- [-D-xylopyranosyl-(12)-]-D-galactopyrano sylcyanidin (5), 3-O-[6-O-(3,4,5-trimethoxycinnamoyl)]-- D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (6), 3-O-[6-O-(3,4-dime- thoxycinnamoyl)]--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (7), 3-O-[(6-O-sinapoyl)--D-glucopyranosyl-(16)--D-galactopyranosyl]cyanidin (8), and 3-O-(-D-galactopyranosyl)cyanidin (9). Except when cinnamic acids were provided in the culture medium, the major anthocyanin present in the two clones examined was 2. When the naturally occurring and some non-naturally occurring cinnamic acids were provided individually in the medium, 1 and 2 were minor components and the anthocyanin acylated with the supplied cinnamic acid, namely 3, 4, 5, 6, or 7 was the major anthocyanin present in the tissue. When caffeic acid was provided the major anthocyanin in the tissue was 4, thereby suggesting that the caffeic acid was methylated before its use in anthocyanin biosynthesis. Other cinnamic acids supplied had limited effects on the anthocyanins accumulated and appeared not to result in the accumulation of new anthocyanins by the tissue. Thus the tissue can use some but not all analogues of sinapic acid to acylate anthocyanins. Additional anthocyanins were detected in extracts of the wild carrot tissue cultures using mass spectrometry (both MS/MS and HPLC/MS). The additional compounds detected have also been found in cultures of black carrot, an Afghan cultivar of Daucus carota ssp. sativa and the flowers of wild carrot giving no evidence for qualitative differences in the anthocyanins synthesized by subspecies, cell cultures from subspecies, or clones from cell cultures. There are major differences in the amounts of individual anthocyanins found in cultures from different subspecies and in different clones from cell cultures. Here anthocyanins without acyl groups were usually found in the tissues and their accumulation is discussed. On the basis of the structures of the isolated anthocyanins, a likely pathway from cyanidin to the accumulated anthocyanins is proposed and discussed.Abbreviations Sin sinapoyl - Fer feruoyl - 4-Coum. 4-coumaroyl - 3,4-MeO₂Cin 3,4-dimethoxyeinnamoyl - 3,4,5-MeO₃Cin 3,4,5-trimethoxycinnamoyl - Cya cyanidin     

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 and characterization of new mutations in the human angiotensinogen gene (AGT)

As part of the multicenter project entitled Pathobiological Determinants of Atherosclerosis in Youth (PDAY), we are testing polymorphisms in candidate genes of atherosclerosis and hypertension for associations with arterial lesions in autopsied young persons. In this study, we used temperature gradient gel electrophoresis (TGGE) to type the Met²³⁵Thr polymorphism in exon 2 of the angiotensinogen gene (AGT) that is associated with essential hypertension in some human populations. In addition to Met²³⁵Thr, we detected and sequenced four other TGGE variants in exon 2 of AGT. These included two new amino acid substitutions (Thr²⁰⁹ Ile and Leu²¹¹Arg) that were found only among black PDAY cases. The frequency of the Ile²⁰⁹ mutation was 0.002 and the frequency of the Arg²¹¹ was 0.006 in 260 black PDAY cases. The other two TGGE variants were Tyr²⁴⁸Cys and a TC substitution at nucleotide position 171 that had been identified in previous studies. We also developed restriction isotyping for rapid typing of each AGT variant using PCR amplification and digestion with diagnostic restriction enzymes.     

Haplotype analysis of familial amyloidotic polyneuropathy

Summary Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant genetic disease characterized by systemic accumulation of amyloid fibrils. A major component of FAP anyloid has been identified as variant transthyretin (TTR, also called prealbumin). In particular, a variant with the substitution ³⁰ValMet has been commonly found in FAP of various ethnic groups. To understand the origin and spread of the ValMet mutation, we analyzed DNA polymorphisms associated with the TTR gene in six Japanese FAP families and several Portuguese FAP patients. Three distinct haplotypes associated with the ValMet mutation were identified in Japanese FAP families, one of which was also found in Portuguese patients. On the other hand, it was found that the ValMet mutation can be explained by a C-T transition at the C_pG dinucleotide sequence of a mutation hot spot. Thus, our findings indicate that the ValMet mutation has probably recurred in the human population, to generate FAP families of independent origin.     

A missense mutation (G197→A) in theα-l-fucosidase gene of fucosidosis patients leads to loss ofα-l-fucosidase

Fucosidosis is an autosomal recessive lysosomal storage disease resulting from the absence of -l-fucosidase activity. Two natural missense mutations (G¹⁹⁷A) and (A⁸⁶⁰G) within the -l-fucosidase gene have been reported to be homozygous in four patients with fucosidosis. Expression of wild-type and mutated -l-fucosidase cDNAs in COS-1 cells revealed complete deficiency of -l-fucosidase for the G¹⁹⁷A transition and a normal level of enzyme for A⁸⁶⁰G. We therefore conclude that the change of G¹⁹⁷A is responsible for fucosidosis in the patients while A⁸⁶⁰G is a normal polymorphic variant of -l-fucosidase.     

Point mutations in the upstream region of the α-galactosidase A gene exon 6 in an atypical variant of Fabry disease

Summary Single point mutations in the upstream region of exon 6 of the -galactosidase A gene were found in two Japanese cases of the cardiac form of Fabry disease; ³⁰¹ArgGln (⁹⁰²GA) in a case that has already been published and ²⁷⁹GlnGlu (⁸³⁵CG) in a new case. They both expressed markedly low, but significant, amounts of residual activity in COS-1 cells. In contrast, two unrelated cases with classic Fabry disease were found to have different point mutations, which showed a complete loss of enzyme activity in a transient expression assay; ³²⁸GlyArg (⁹⁸²GA) in the downstream region of exon 6 in one case and two combined mutations, ⁶⁶GluGln (¹⁹⁶GC)/¹¹²ArgCys (³³⁴CT), in exon 2 in the other. We conclude, on the basis of the results recorded in this study and those in previous reports, that the pathogenesis of atypical Fabry disease is closely associated with point mutations in the upstream region of exon 6 of the -galactosidase A gene.     

A new translocation t(1;4;11) in congenital acute nonlymphocytic leukemia (acute myeloblastic leukemia)

Summary A new translocation t(1;11;4)(1pter1p32::11q23 11q13::4p164qter) was found in the peripheral blood of a patient with congenital acute myeloblastic leukemia (AML). It was concluted that this translocation may represent a new mutation, which caused the leukemia with very high leukocytosis, hepatosplenomegaly, leukemic infiltration of the majority of the organs, and a very poor prognosis.     

Confirmation of regional assignment of nucleoside phosphorylase (NP) on chromosome 14 by gene dosage studies

Summary Gene dosage studies yielded results consistent with assignment of the locus for nucleoside phosphorylase to band 14q13. The red blood cells from a patient with the karyotype 47,XX,+der(14),t(8;14)(8qter8q24: :14q2114pter)pat had enzyme activity 50% higher than red cells from 47 normal controls, two trisomies involving chromosomes other than 14, and five balanced translocations involving chromosome 14. On the other hand, the red cells of a case with a karyotype 45,XX,-14,-22,+der(22),t(14;22)(14qter14q11 or 14q12::22p1122qter)mat and a case with a karyotype 47,XX, +der(14),t(14;16)(14pter14q11::16q2416qter)mat had normal activity.     

Negative-ion fast atom bombardment tandem mass spectrometry for characterization of sulfated unsaturated disaccharides from heparin and heparan sulfate

Negative-ion fast atom bombardment tandem mass spectrometry has been used in the characterization of non-, mono-, di- and trisulfated disaccharides from heparin and heparan sulfate. The positional isomers of the sulfate group of monosulfated disaccharides were distinguished from each other by negative-ion fast atom bombardment tandem mass spectra, which provide an easy way of identifying the positional isomers. This fast atom bombardment collision induced dissociation mass spectrometry/mass spectrometry technique was also applied successfully to the characterization of di- and trisulfated disaccharides.Abbreviations FABMS fast atom bombardment mass spectrometry - CID collision induced dissociation - MIKE mass analysed ion kinetic energy - MS/MS mass spectrometry/mass spectrometry - HPLC high performance liquid chromatography - UA d-gluco-4-enepyranosyluronic acid - CS chondroitin sulfate - DS dermatan sulfate - HA hyaluronan - Hep heparin - HS heparan sulfate - UA(14) GlcNAc 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNAc 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcN6S 2-amino-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcN 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcN6S 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcNS 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNS 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(13)GalNAc 2-acetamido-2-deoxy-3-O-(-d-Gluco-4-enepyranosyluronic acid)-d-galatose - UA(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA2S(13)GalNAc 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-galactose - UA2S(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA2S(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA(13)GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose - UA(13)GlcNAc 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose.     

Monoclonal antibody specific for α2→8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing 28-linked polysialic acid (8Neu5Gc2) _n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (8KDN2) _n 6(KDN23Gal13GlNAc13) GalNAc1 residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.kdn8kdn was shown specifically to recognize the 28-linked oligo/polyKDN sequences, (8KDN2) _n , and to be able to distinguish specifically (8KDN2) _n chains from (8Neu5Ac2) _n and (8Neu5Gc2) _n chains. The antibody was used successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several controls verified the specificity of the immunohistochemical staining, thus providing the first demonstration of (8KDN2) _n sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (8KDN2) _n chains and will facilitate studies on their biosynthesis, intracellular localization and function.     

Possible complex translocation t(9; 14; 13)(q12;pl?;q31) in mother of a child with 9p-trisomy syndrome

Summary A mentally retarded boy with trisomy 9p is described. This trisomy arose through aberrant segregation of translocation chromosome during meiosis in his mother, who has a complex translocation involving chromosomes 9, 13, and 14. Based on both G-, Q-banding, and DNA replication patterns, the patient's karyotype was identified as 47,XY,-13, +(9;13) (9pter9q12::13q3113qter), +t(13;14) (13pter13q31::14pl?14pter). We suppose his mother's karyotype to be 46,XX,-9,-13,-14,+t(9;13) (9pterq12::13q3113qter), +t(13;14) (13pter13q31::14pl?14pter), +t(9;14) (9qter9q12::14pl?14qter). His phenotypically normal brother and sister are also carriers, having the same translocation chromosome as their mother. Clinical findings of the patient included peculiar face with hypertelorism, prominent nasal bridge and deformed helix, marked delay of osseous development, hypoplastic phalangia in fingers and toes, dysplastic nails and absence of digital triradii.     

Possible intrachromosomal duplication in a case of trisomy 9p

Summary A 5-year-old boy with multiple minor anomalies and mental retardation was found to have chromosomal condition of 46,XY,inv dup(9p)(pterp13::p21p24::p13qter). The clinical features of the propositus fit well with those of trisomy 9p which have been established to be a clinical entity.     

Mapping of the regulatory type Iα and catalytic β subunits of cAMP-dependent protein kinase and interleukin 1α and 1β in the pig

The genes coding for the regulatory type I subunit (PRKAR1A) and the catalytic subunit (PRKACB) of cAMP-dependent protein kinase and the genes for interleukin 1 (IL1A) and interleukin 1 (IL1B) were localized in the pig by means of radioactive in situ hybridization. PRKAR1A was mapped to 12p1.4 and PRKARB to 6q3.1 q3.3. The genes for IL1A and IL1B were both assigned to Chromosome (Chr) 3, in the region q1.2 q1.3 and q1.1 q1.4, respectively. The cDNA nucleotide sequences of these porcine genes were compared with those of human, mouse, and cattle. The location of the genes was discussed in relation to the position of their homologous loci in these mammalian species.     

Structural study of fucoidan from Cladosiphon okamuranus tokida

A structural study was carried out on a fucoidan isolated from the brown seaweed Cladosiphon okamuranus. The polysaccharide contained fucose, glucuronic acid and sulfate in a molar ratio of about 6.1 : 1.0 : 2.9. The results of Smith degradation showed that this polysaccharide has a linear backbone of 13-linked -fucopyranose with a half sulfate substitution at the 4-positions, and a portion of the fucose residues was O-acetylated. The data obtained from partial acid hydrolysis, a methylation analysis and NMR spectra indicated that the -glucuronic acid residue is linked to the 2-positions of the fucose residues, which were not substituted by a sulfate group. These results indicated that the average structure of this fucoidan is as follows: -[(3Fuc-4(±OSO₃-)1–)₅3[GlcA12]Fuc1–]_n–. (Half of each fucose residue was sulfated. One O-acetyl ester was present in every 6 fucose residues.)