A Gln-281 to Arg substitution in α-l-fucosidase is responsible for a common polymorphism detected by isoelectric focusing

A common polymorphism of human -l-fucosidase consists of three phenotypes (Fu 1, Fu 2, and Fu 2-1) assigned by isoelectric focusing. The phenotypes are determined by two codominant alleles (Fu¹ and Fu²). Isozymes with the Fu 2 phenotype have more basic pI than Fu 1, while Fu 2-1 is a mixture of Fu 2 and Fu 1. Recently, a missense mutation (A⁸⁶⁰G) in the -l-fucosidase gene was described that did not affect -l-fucosidase activity. The mutation causes the substitution of Arg (pKa^Guan=12.5) for Gln-281, which has no ionizable side chain. Isoelectric focusing profiles of extracts of COS-1 cells transfected with wild-type or mutant -l-fucosidase cDNAs had phenotypes of Fu 1 and Fu 2, respectively. Next, 20 human lymphoid cell lines were examined for the occurrence of the A⁸⁶⁰G mutation and expression of the Fu 1, Fu 2, and Fu 2-1 phenotypes. Eight lines with Fu 2 were homozygous for the A⁸⁶⁰G mutation; six lines with Fu 1 were homozygous for the normal nucleotide (A⁸⁶⁰); and six lines with Fu 2-1 were heterozygous. Thus, the A⁸⁶⁰G mutation is the molecular basis for the protein phenotypes and the Fu¹ and Fu² alleles. The normal nucleotide (A⁸⁶⁰) is responsible for Fu¹ and the A⁸⁶⁰G mutation for Fu².     

Effects of irreversible and reversible cholinesterase inhibitors on single acetylcholine-activated channels

Summary The use of cholinesterase (CHE) inhibitors provided valuable information about the mechanism(s) of neuromuscular transmission, but questions on side effects at the level of AChactivated channels were raised. Patch-clamp recording was used to study the effects of prostigmine (PST) and methanesulfonyl fluoride (MSF), a reversible and an irreversible cholinesterase inhibitor, respectively, on ACh-activated channels. We found that these drugs diminish the average dwell time of elementary currents from around 5 msec (control) to less than 1 msec in the presence of PST (20 m) or MSF (5 mm) (at room temperature). With MSF the ACh-activated channel conductance of the most frequently observed amplitude class decreased from 45 pS (control) to 30 pS, but not in the presence of PST. In control conditions there were also amplitude classes of 60 and 24 pS, with probabilities of occurrence <10%. In the presence of 1.5 mm MSF, where current dwell time was not affected, additional subconductance states of 19 and 36 pS were observed and may be due to partial blockade of the open channel. We conclude that the drug of choice to be used in studies on the role of CHE in the neuromuscular transmission is MSF, because at 20 m PST, where blockade of ACh-activated channels is significant, cholinesterase was reported to be partially inhibited, whereas at 1 mm MSF it is fully inhibited and the dwell time of ACh-activated channels is not affected.This research was supported by the Ministry of Science and Technology of Republic of Slovenia. M.S. would like to acknowledge the Italian Board of Education (M.P.I.) for support. The analog-to-digital converter (CED 1401) was kindly donated by The Wellcome Trust.     

Zur Genetik des Lp-Systems

A new method — the precipitating electrophoresis in an Anti-Lp serum containing agar medium — is applied to the investigation of 527 sera from apparently healthy blood donors and a series of 78 families with altogether 203 children for Lp(a). The technique is described in detail, of which the most important features are outlined. Parallel tests in Ouchterlony's double diffusion technique are carried out. By comparison of the distribution of phenotypes Lpa⁺⁺, Lpa⁺, Lpa(+) and Lpa — among the blood donors and the children, a significant difference is found. This difference is due to a trend of fetal selection of Lpa — positives in heterologous matings. In addition, the factor Lpa seems to influence the fertility in these matings negatively, while no distinct differences in the age distribution among men, women, girls and boys are detected. A close correlation between the expression of phenotypes among the parents and their related children gives evidence for the inherited regulation of the quantitative level of Lp phenotypes. Only few exceptions from this rule do occur. These findings cannot be explained by methodical reasons alone. The inheritance of quantitative differences of Lpa leads to the assumption of either a genetic model of a row of alleles where the regulation of the higher concentrations of the Lp protein must be dominant over the regulation of lower concentrations and the Lp negatives, or a multifactorial system. A decision cannot be made basing on the here reported findings.

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Direktor: Prof. Dr. H. Elbel)

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Herrn Professor Dr. med. H. Elbel zur Vollendung des 60. Lebensjahres gewidmet.     

Genetically polymorphic α-l-fucosidase (FUCA1) isozymes detected in blood plasma

The main isozyme patterns of desialylated blood plasma or serum -l-fucosidase (FUCA) were found to be almost identical to those of semen, urine, placental extracts, and leukocyte lysates, when detected by polyacrylamide gel isoelectric focusing, and activity staining using the fluorogenic substrate 4-methylumbelliferyl--l-fucopyranoside. Three phenotypes (1, 2-1, and 2) determined from plasma samples were identical to the phenotypes from urine and leukocyte lysates from the same individuals. A population study of plasma samples collected from 485 Japanese individuals indicated that the frequencies of the FUCA11 ^* and FUCA12 ^* alleles were 0.7505 and 0.2495, respectively. The mean plasma enzyme activities (+SD) of the three phenotypes were 318.8 ± 116.7 nmol/ml per h for type 1, 268.0 ± 108.3 nmol/ml per h for type 2-1, and 233.2 ± 84.4 nmol/ml per h for type 2. The mean activities of types 1 and 2 suggest that, on average, the FUCA11 ^* gene product in plasma has about 1.4 times the activity of FUCA12 ^*.     

Possible influence of BCHE locus of butyrylcholinesterase on stature and body mass index

Butyrylcholinesterase activity has been shown to be positively associated with weight and body mass index (BMI). The present study was carried out to search for an association between variants of the BCHE gene and weight, stature, and BMI on the basis of means and variances compared between nonusual variants and their respective usual controls. Individuals bearing the atypical mutation (N = 52) did not differ from their usual phenotype controls (N = 104) in these parameters. The BCHE*U/BCHE*K individuals (N = 222) presented a significantly higher BMI variance than their BCHE*U/BCHE*U controls (N = 222, F = 1.40, P = 0.012). This higher BMI variance does not seem to be an isolated effect of the K mutation, but appears to be the result of an interaction between the K allele and the usual allele, since no such difference in variance was detected between BCHE*K/BCHE*K individuals (N = 23) and their BCHE*U/BCHE*U (N = 23) controls. These data may suggest a relation between variability in the BCHE locus itself and BMI. Individuals with the BCHE UF phenotype (N = 45) showed a significantly higher mean stature (about 3 cm more; P = 0.02) than their controls with the usual phenotype (N = 135). A role in cell proliferation has been proposed for BCHE, and since growth depends on the number of mitoses, it is not unexpected that variants of this enzyme may influence body stature in different ways. This study reports the first data on the relation of BCHE alleles to anthropometric characters.     

The xylogalactan sulfate from Chondria macrocarpa (Ceramiales,Rhodophyta)

A structure is proposed for the complex xylogalactan sulfate from Chondria macrocarpa. The hot-water extract of C. macrocarpa was desulfated or alkali-treated and Smith degraded. Constituent sugars and their substitution patterns were identified using a modified Hakamori methylation procedure suited to sulfated polysaccharides and a double hydrolysis-reduction protocol that yielded derivatives from all of the sugar residues, including the labile 3,6-anhydrogalactosyl residues. The polymer has an agar-type backbone of alternating 3-linked \-d- and 4-linked -L-galactopyranosyl units. The d-residues are partially sulfated on O-2 (50%) and O-6 (20–30%). About 40% of the l-residues are present as the 3,6-anhydride and 25% as its precursor l-galactose 6-sulfate. A significant proportion of the remaining l-galactosyl residues have both a d-xylopyranosyl substituent on O-3 and a sulfate ester on O-6 and are stable to alkali.     

Binding of concanavalin A and wheat germ agglutinin by murine peritoneal macrophages: ultrastructural and cytophotometric studies

Summary Concanavalin A and wheat germ agglutinin were employed in conjunction with the horseradish peroxidase-diaminobenzidine method for the detection of sugar residues on the surface coat of exudate and resident murine peritoneal macrophages. Electron microscopical and cytophotometric techniques were used for the visualization and quantification of the final reaction product on the surface of cells. After incubation with concanavalin A and wheat germ agglutinin, both exudate and resident macrophages showed readily detectable final reaction product indicating the presence of numerous, easily accessible, -methyl-d-mannosyl andN-acetyl-d-glucosaminyl residues on their surface. The binding of concanavalin A was higher with resident than with exudate macrophages. With wheat germ agglutinin, a different pattern of lectin binding was observed: more electron-dense product was deposited on exudate than on resident macrophage surfaces. The binding of concanavalin A and wheat germ agglutinin to macrophages was inhibited by the competing sugars -methyl-d-mannoside andN-acetyl-d-glucosamine, respectively.     

Nomenklatur,Chromosomenzahlen und Evolution vonArabis auriculata Lam.,A. nova Vill. undA. verna (L.) R.Br. (Brassicaceae)

Zusammenfassung Auf Grund des Holotypus im Herbarium P-LA wird der NameArabia auriculata Lam. als korrekter Name für die am weitesten verbreitete Species der SectionAlomatium DC. emend. O. E.Schulz wieder eingeführt, als deren Typusart sie zugleich vorgeschlagen wird.A. auriculata Lam. 1783 wurde in letzter Zeit fälschlich in europäischen Floren mit dem jüngeren Synonym A. recta Vill. 1789 belegt und in vorderasiatischen Floren A. nova Vill. 1779 genannt.A. nova Vill. ist jedoch eine europäisch-submediterrane Art, die nicht mitA. auriculata Lam. conspecifisch ist. A. auriculata undA. nova sind diploid (2 n=16),A. verna ist tetraploid (2 n=32, diese Zahl wird erstmals veröffentlicht). A. auriculata undA. nova sind vorwiegend autogam,A. verna ist ebenfalls selbstkompatibel und trotz ihrer auffälligeren Blüten fakultativ autogam. Die drei wärmeliebenden Arten sind normalerweise einjährig, manchmal jedoch auch zweijährig. Als Einjährige sind sie in der Gattung als abgeleitet anzusehen.

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Nomenclature, chromosome numbers and evolution ofArabis auriculata Lam.,A. nova Vill., andA. verna (L.) K.Br. (Brassicaceae)

Summary Based on its holotype in the herbarium P-LA the nameArabis auriculata Lam. 1783 is re-established as the correct name of the widespread annual species, which was falsely called A. recta Vill. 1789 in recent European floras, and A. nova Vill. 1779 in recent floras of Western Asia. It is shown that A. nova Vill. refers to a European species of the submediterranean region, which is not conspecific withA. auriculata Lam. A. auriculata Lam. is proposed to be the lectotype ofArabis L. sect.Alomatium DC. emend. O. E.Schulz, comprising the species discussed here. A. auriculata andA. nova are diploids (2 n=16),A. verna is a tetraploid (2 n=32); the latter chromosome number is reported here for the first time. A. auriculata andA. nova are predominantly self-fertilizing,A. verna (though having more conspicuous flowers) is self-compatible and thus facultatively autogamous as well. The three thermophilous species are normally annual, only sometimes biennial. As annuals they are to be regarded as evolutionary derived within the genusArabis.

     

Protein reactions with methyl and ethyl vinyl sulfones

Disulfide bonds of bovine serum albumin and wool were reduced byn-tributylphosphine to sulfhydryl groups that were then modified by methyl or ethyl vinyl sulfone in a nucleophilic addition reaction toS-(-ethylsulfonylmethyl)-l-cysteine andS(-ethylsulfonylethyl)-l-cysteine, respectively. The reductive alkylation was carried out either simultaneously, with both the reducing and alkylating agents present in the reaction mixture, or sequentially, with the reduced proteins first isolated before alkylation. Amino acid analysis studies showed that authentic, syntheticS-(-ethylsulfonylethyl)-l-cysteine eluted as a well-resolved peak after serine but that the peak associated with the corresponding methyl derivative overlapped the corresponding peak due to threonine. The extent of alkylation of the sulfhydryl groups of cysteine, -NH₂ of lysine, and NH groups of the imidazole ring of histidine was also measured by amino acid analysis. The results show that alkyl vinyl sulfones have a strong chemical affinity for protein functional groups.     

Butyrylcholinesterase Genetic Variants: Association with Cocaine Dependence and Related Phenotypes

Objective

The search for genetic vulnerability factors in cocaine dependence has focused on the role that neuroplasticity plays in addiction. However, like many other drugs, the ability of an individual to metabolize cocaine can also influence susceptibility to dependence. Butyrylcholinesterase (BChE) metabolizes cocaine, and genetic variants of the BChE gene (BCHE) alter its catalytic activity. Therefore, we hypothesize that cocaine users with polymorphisms in BCHE can show diverse addictive behaviors due to differences in effective plasma concentrations of cocaine. Those polymorphisms might also influence users to prefer one of the two main preparations (crack or powder cocaine), despite having equal access to both. The present work investigates polymorphisms in BCHE and if those genetic variants constitute risk factors for cocaine dependence and for crack cocaine use.

Methods

A total of 1,436 individuals (698 cocaine-dependent patients and 738 controls) were genotyped for three single nucleotide polymorphisms (SNPs) in BCHE: rs1803274, rs4263329, and rs4680662.

Results

For rs4263329, a nominal difference was found between cases and controls. For rs1803274 (the functional SNP), a statistically significant difference was found between patients who used crack cocaine exclusively and those who used only powder cocaine (P = 0.027; OR = 4.36; 95% CI = 1.18–16.04). Allele frequencies and genotypes related to other markers did not differ between cases and controls or between the two cocaine subgroups.

Conclusions

Our findings suggest that the AA genotype of rs1803274 is a risk factor for crack cocaine use, which is more addictive than powder cocaine use. Further studies are needed in order to confirm this preliminary result and clarify the role of BCHE and its variants in cocaine dependence.     

A dual staining method for neutral complex carbohydrates using alkaline phosphatase-labeled concanavalin a and periodic acid-Schiff

Summary A method has been developed for the dual staining of neutral complex carbohydrates in light microscopy. It combines an alkaline phosphatase-labeled concanavalin A-5-bromo-3-indolyl phosphate, p-toluidine salt (Con A-ALP-BIPT) method with periodic acid-Schiff (PAS) sequence. With the present dual staining method, it is possible to color -d-glucosyl and -d-mannosyl residues blue and 1,2-glycol groups of neutral complex carbohydrates magenta. The validity of this method has been confirmed with appropriate histochemical controls and enzyme digestions on test tissues.     

Concanavalin A-peroxidase-diaminobenzidine-periodic acid-m-aminophenol-fast black salt K: a method for the dual staining of neutral complex carbohydrates.

Synopsis A method has been developed for the dual staining of neutral complex carbohydrates in light microscopy. It combines a concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB) method with a period acid-m-aminophenol-Fast Black salt K (PA-AP-FBK) sequence. With the combined method it is possible to stain -D-glycosyl and -D-mannosyl residues brown and 1,2-glycol groups of neutral complex carbohydrates blackish purple. The validity of the method has been confirmed with appropriate histochemical controls and enzyme digestions on test tissues.     

A quantitative cytochemical assay of β-galactosidase in single cultured human skin fibroblasts

Summary A quantitative cytochemical method for the measurement of -galactosidase activity in cultured human skin fibroblasts has been developed using 5-bromo-4-chloro-3-indolyl--D-galactopyranoside as the indigogenic substrate. The method relies upon the oxidation of the primary reaction product by ferro/ferricyanide during which an insoluble indigo dye is generated as the final reaction product. The reaction was linear with time up to 60 min using the final cytochemical standard procedure. The enzyme showed maximum activity at pH 4.0 to 4.1. The concentration optima of indigogenic substrate and potassium ferro/ferricyanide were 3.67 mM and 3.13 mM respectively. The presence of sodium chloride activated -galactosidase up to 100 mM, but was inhibitory above that concentration. The enzyme was inhibited by N-ethylmaleimide, N-acetyl-D-galactosamine and heparin. The enzyme molecules were shown to diffuse out of the cells using media without a suitable inert colloid stabilizer. However, diffusion was completely prevented by using polyvinyl alcohol (PVA) grade G18/140. Air-drying of cells was essential to make the cell membrane permeabel to the substrate and, thereby, to avoid a pronounced lag phase. However, in a biochemical analysis, air-drying itself caused a decrease in enzyme activity to 43% of the control. Even after air-drying lysosomal latency could still be demonstrated by using PVA grade G04/140.Control persons, one carrier of and two patients with -galactosidase deficiency were easily identified as belonging to three separate groups by using the cytochemical assay.It is proposed that the quantitative cytochemical approach may also be applied to cultured human amniotic fluid cells or chorion biopsies giving a rapid prenatal diagnosis of -galactosidase deficiency due to the small number of cells needed in the analysis.     

Displacement of excitatory amino acid receptor ligands by acidic oligopeptides

A number ofD-glutamyl andL-aspartyl dipeptides, glutathione, -D-glutamylglycine and -D-glutamyltaurine, were tested for their efficacy to displace ligands specific for different subtypes of excitatory amino acid receptors from rat brain synaptic membranes. In general, theL enanthiomorphs of -glutamyl peptides were more potent displacers than -D-glutamylglycine and-taurine but the latter were more specific for the quisqualate type of receptors. -L-glutamyl-L-glutamate was the most effective dipeptide in displacing the binding of glutamate, 2-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) and 2-amino-5-phosphonoheptanoate (APH), whereas -L-glutamyl-L-aspartate was the most effective in the binding of kainate. Both oxidized and reduced glutathione were inhibitory, being most potent in the binding of AMPA. -L-Glutamylaminomethylsulphonate was most effective in the binding of APH. The most potent -L-glutamyl peptides (glutathione, -L-glutamyl-L-glutamate,-L-aspartate, and-glycine) may act as endogenous modulators of excitatory aminoacidergic neurotransmission.     

Structural analysis ofBacillus licheniformis 86 surfactant

Summary A tentative structure and composition of a surfactant, BL-86, produced byBacillus licheniformis 86 is described. The surfactant is a mixture of lipopeptides with the major components ranging in size from 979 to 1091 Da and varying in increments of 14 Da. The variation in molecular weight represents changes in the number of methylene groups in the lipid and/or peptide portion of the surfactant. There are 7 amino acids per molecule. The peptide portion is composed of the following amino acids: glutamic acid or glutamine (glx), aspartic acid or asparagine (asx), valine, leucine, and isoleucine at a ratio of 1.01.01.43.00.6, respectively. The leucine is present as both thed andl isomers at a ratio of about 21, respectively. Forty percent of the molecules containl-valine instead ofl-isoleucine. The glx and asx are present as a combination ofl-glutamic acid andl-asparagine and/orl-glutamine andl-aspartic acid. The N-terminus of the peptide is blocked, most likely by a peptide bond to the lipid portion. An ester carbonyl structure is present, which could be a part of a lactone ring connecting the position of the lipid to one of the carboxyl groups in the peptide. The lipid portion is composed of, on average, 8–9 methylene groups, and contains a mixture of linear and branched tails. Results of DCI-MS and FAB-MS analyses, as well as surface tension measurements, of purified BL-86 HPLC fractions support the proposed composition.     

Lactose utilization byVibrio vulnificus

The ability of wild-type strains ofVibrio vulnificus to utilize lactose as a sole source of carbon and energy and produce acid in lactose-containing media is associated with the appearance of spontaneous lactose-utilizing mutants. These contain increased activities of an enzyme able to hydrolyzeo-nitrophenyl--d-galactoside as well as lactose. This activity is constitutive in some mutants and inducible by both lactose and isopropyl--d-thiogalactoside in others. A limited survey of otherVibrio species indicates thatV. pelagius also can acquire, by mutation, the ability to grow on and make acid from lactose. No immunological cross-reaction was detected between the enzymes fromVibrio and the -galactosidases ofEscherichia coli andKlebsiella.     

On the gas vacuoles of the halobacteria

Summary The cells of Halobacterium sp., strain 5, contain a large number of highly refractile bodies of the type which Petter (1932) suggested were gas-filled vacuoles. The present studies support Petter's contention, but the evidence for the exact chemical nature of the vacuole content is still indirect. It is not carbon dioxide or oxygen, but might possibly be nitrogen. Strain 5 loses spontaneously and with a high frequency the ability to make the vacuoles.When vacuolated cells are subjected to pressure, the vacuoles disappear, but can recover upon aeration. Oxygen and the organic constituents of the growth medium stimulate the recovery, whereas 2.4-dinitrophenol inhibits it. A procedure is described for the isolation of the vacuoles. The vacuoles are bounded by a membrane which reveals itself in electron micrographs of thin sections as a 1-layered structure about 30 Å thick.Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday.     

Method for the synthesis of phosphinic acids from hypophosphites V. The synthesis of pseudo-α,α-dipeptides

Summary. Neurolathyrisim is a motor neuron disease characterized by spastic paraparesis in the hind legs, and is caused by grass pea, Lathyrus sativus, which contains the excitotoxic amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (L--ODAP), an -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamatergic receptor agonist. In an attempt to make a useful model of this disease, the CNS distribution and toxicity of L--ODAP was studied in rat neonates after parenteral administration. L--ODAP was detected in the spinal cord as well as in the pons/medulla oblongata, though only small amounts in the latter. Repeated injection of L--ODAP resulted in rats with paraparesis of the legs, though at a low incidence rate of 0.032. These paralyzed rats displayed the severe atrophy of the ventral root of the lumbar cord as well as degenerations of motor neuron. The rats were useful models for the study of motor neuron degeneration in the spinal cord.     

Differences in the distribution of β-glucuronidase activity demonstrated with the Fishman-Baker method in the adrenal cortex of albino rats treated with corticosteroids and after hypophysectomy

Summary Histohemical estimation of -glucuronidase was performed in the adrenals of normal rats, rats treated with corticosteroids and hypophysectomized rats. In these experiments the method described by Fishman and Baker was used. A difference was found between normal rats and rats treated with hydrocortisone and corticosterone on the one hand and hypophysectomized animals on the other. In the first group a positive reaction was found in the zona intermedia, the zona fasciculata, and the zona reticularis. In the second group a positive reaction was found in the male animals exclusively in the zona intermedia and in the female animals in both the zona intermedia and the zona glomerulosa. Although the distribution of the blue precipitate in the adrenal cortex of untreated rats is similar to the distribution of -glucuronidase as determined histo-biochemically by Nayyar and Glick we doubt, in spite of the observed difference, that the Fishman-Baker method is specific for -glucuronidase. Replacement of the specific substance 8-hydroxyquinoline glucuronide by 8-hydroxyquinoline did not affect the results.     

Induction of extracellular arabinases on monomeric substrates in Aspergillus niger

The induction of extracellular arabinases by pentose sugars and polyols generated by the metabolic pathway of l-arabinose and d-xylose catabolism in Aspergillus niger was investigated. Induction occurred with l-arabinose and l-arabitol but not with d-xylose or xylitol. l-arabitol in particular was found to be a good inducer for -l-arabinofuranosidase and endo-arabinase activities. Western blotting analysis showed both -l-arabinofuranosidase A and B to be present. No induction was observed using d-arabitol. Unlike the wild type A. niger N402 strain, the A. niger xylulose kinase negative mutant N572 also showed induction of -l-arabinofuranosidases A and B and endo-arabinase activity on d-xylose and xylitol. This is due to metabolic conversion of these compounds leading to the accumulation of both xylitol and l-arabitol in this mutant, the latter of which then acts as inducer. The induction of the two -l-arabinofuranosidases and endo-arabinase is under the control of two regulatory systems namely pathway specific induction and carbon catabolite repression. Under derepressing conditions in the wild type only -l-arabinofuranosidase B could be detected by Western blotting analysis. This indicates that -l-arabinofuranosidase B is of importance in the initiation of specific induction of the various arabinose activities in A. niger grown on arabinose containing structural polysaccharides.Abbreviations PNA p-nitrophenyl--l-arabinofuranoside