Multiple mutations are responsible for the high frequency of metachromatic leukodystrophy in a small geographic area.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. The disease occurs panethnically, with an estimated frequency of 1/40,000. Metachromatic leukodystrophy was found to be more frequent among Arabs living in two restricted areas in Israel. Ten families with affected children have been found, three in the Jerusalem region and seven in a small area in lower Galilee. Whereas all patients from the Jerusalem region are homozygous for a frequent mutant arylsulfatase A allele, five different mutations were found in the families from lower Galilee. In patients of Muslim Arab origin, we have found a G86-->D, a S96-->L, and a Q190-->H substitution. Two different defective arylsulfatase A alleles, characterized by a T274-->M and a R370-->W substitution, respectively, have been found among the Christian Arab patients. All mutations were introduced into the wild-type arylsulfatase A cDNA. No enzyme activity could be expressed from the mutagenized cDNAs after transfection into heterologous cells. In all instances, the patients were found to be homozygous for the mutations, and four of the five mutations occurred on different haplotypes. The clustering of this rare lysosomal storage disease in a small geographic area usually suggests a founder effect, so the finding of five different mutations is surprising.     

Determination of N-(9-( -D-ribofuranosyl)purin-6-ylcarbamoly)threonine at the picomole level in transfer-RNA

An assay has been developed for quantitation of the modified nucleoside, t⁶A, in tRNA at the pmole level. For tRNA from a variety of species, the content of t⁶A was found to be 0.18–0.25 mole %. These values lend support to the suggestion that t⁶A is located at the 3′-end of the anticodon in tRNA's whose codons begin with adenosine. Essentially no t⁶A was found in Mycoplasma sp. (Kid) tRNA which is deficient in many modified nucleosides. In the rat, no organ specific differences were found. The amount of t⁶A in Novikoff hepatoma tRNA was essentially the same as in tRNA from normal rat liver.     

Helminths: comparison of their rhodoquinone

A rhodoquinone (RQ) has been isolated from Stephanurus dentatus and has been shown to be identical with RQ-9 from Ascaris lumbricoides var. suis (A. suum). The ratio of RQ/mg N is fairly constant among larval, juvenile, and adult forms of S. dentatus with an average value of 1.63 μg/mg N. A Chromatographie survey found RQ in all eleven species of helminths tested. Ubiquinone (UQ) was found in all larval forms, a free living nematode (Rhabditis strongyloides) and helminths with prepatent periods shorter than 35 days.     

Distribution and 16S rDNA sequences of <Emphasis Type="Italic">Argas monachus</Emphasis> (Acari: Argasidae), a soft tick parasite of <Emphasis Type="Italic">Myiopsitta monachus</Emphasis> (Aves: Psittacidae)

Specimens of Argas monachus Keirans et al. were collected from Myiopsitta monachus nests in 42 localities in Argentina and Paraguay from 2006 to 2010. A list of localities where this tick has been found is presented. 16S rDNA sequences of specimens of A. monachus from different localities were compared to confirm whether they belong to the same specific taxon. Argas monachus is present in the phytogeographic provinces of Chaco, Espinal, and Monte, but not in the Pampa (all from de Chaco Domain) where the host is well distributed. No differences were found among 16S rDNA sequences of geographically distant specimens.     

Some characteristics of new tissue-binding proteins for metabolites of vitamin D other than 1,25-dihydroxyvitamin D

Protein(s) have been found in a wide range of tissues which have a high affinity for 25-hydroxycholecalciferol. Of the tissues examined only erythrocytes do not have this protein. The properties of the protein have been examined and it has been found that the association constants range from 2 · 10⁹ to 5 · 10⁹ M⁻¹ and the sedimentation constants between 5.0 and 6.0 S. It was not possible to distinguish the proteins from the different tissues by their S values, mobility on gel electrophoresis or behaviour on ion-exchange chromatography. These techniques were all used, however, to show that the tissue 25-hydroxycholecalciferol binding protein is distinct from the main plasma binding protein for this steroid and from the intestinal 1,25-dihydroxycholecalciferol-binding protein. A protein has been found in the plasma of rachitic animals but not of normals, which is apparently indistinguishable from this new tissue 25-hydroxycholecalciferol-binding protein. The steroid specificity of this new binding protein has been shown to be dependent upon a C-25 hydroxyl group, and an intact conjugated double bond system. Possible functions for this protein have been briefly discussed.     

Humanisation and characterisation of PR1A3, a monoclonal antibody specific for cell-bound carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is highly expressed by most tumours of gastrointestinal origin, but its use as a target for tumour therapy is complicated by the high levels of soluble CEA that are found circulating in the blood of cancer patients. A monoclonal antibody PR1A3 has been prepared, which binds preferentially to cell-surface rather than soluble CEA, this cell selectivity should make PR1A3 an ideal candidate for antibody-targeted tumour therapy. PR1A3 has been humanised and shown to retain its cell-surface specificity and affinity. Stable expression of the humanised antibody from chinese hamster ovary (CHO) cells has been achieved after transfection and amplification. Since PR1A3 binds preferentially to cell-associated CEA, a cell-free enzyme-linked immunosorbent assay (ELISA) has been developed to allow characterisation and routine assay of the antibody. This assay was developed using a recombinant chimeric protein constructed by cloning the domain of CEA that is bound by PR1A3 (the B3 domain) into a hybrid gene containing the Fc portion of IgG and three domains of biliary glycoprotein. Stable expression of this hybrid protein has been achieved from CHO cells. In ELISA both humanised and murine PR1A3 bound strongly to this antigen but only at a minimal level to soluble CEA. Two binding sites for the antibody were found on the gastric carcinoma cell line MKN45, one of higher affinity (1 nM) and the other at lower affinity (60 nM). Similar affinities were found for both murine and humanised antibodies. The data presented make it unlikely that the differential binding to cell-surface as distinct from soluble CEA can be accounted for by low affinity of PR1A3 for CEA, and provides further support for the hypothesis that some conformational change takes place on CEA release from cells and that it is this change that blocks PR1A3 binding to its epitope. Received: 5 October 1998 / Accepted: 19 November 1998     

Identification of the Phosphorylated Residues in TveIF5A by Mass Spectrometry

The initiation factor elF5A in Trichomonas vaginalis （TvelF5A） is previously shown to undergo hypusination, phosphorylation and glycosylation. Three different pI isoforms of TvelF5A have been reported. The most acidic isoform （pI 5.2） corresponds to the precursor TvelF5A, whereas the mature TvelF5A appears to be the most basic isoform （pI 5.5）. In addition, the intermediary isoform （pI 5.3） is found only under polyamine-depleted conditions and restored with exogenous putrescine. We propose that differences in PI are due to phosphorylation of the TvelF5A isoforms. Here, we have identified phosphorylation sites using mass spectrometry. The mature TvelF5A contains four phosphorylated residues （$3, T55, T78 and T82）. Phosphorylation at $3 and T82 is also identified in the intermediary TvelF5A, while no phosphorylated residues are found in the precursor TvelF5A. It has been demonstrated that elF5A proteins from plants and yeast are phosphorylated by a casein kinase 2 （CK2）. Interestingly, a gene encoding a protein highly similar to CK2 （TvCK2） is found in T. vaginalis, which might be involved in the phosphorylation of TvelF5A in T. vaginalis.     

Araucnephia iberaensis n. sp., a neotropical black fly with a peculiar distribution (Diptera-Simuliidae)

Araucnephia Wygodzinsky & Coscarón is a Neotropical black fly genus in which only one species from Central Chile is known. Another species has now been found in Corrientes province on the eastern side of the Iberá tropical swamps of Argentina, on the western border of the mountainous region of southern Brazil. This new species, A. iberaensis, is herein described and illustrated and information on its bionomics is recorded. It is an interesting species because previous to its discovery no black fly genus or subgenus from Central Chile region has been found in tropical areas, because these two regions are separated by the Monte and Pampas realms. Similarly, no Brazilian genus or subgenus has crossed the Andes mountains to Chile. A comparison with other Neotropical, Nearctic, Ethiopian (Afrotropical) and Australian Prosimuliini (sensu Crosskey & Howard) showed Araucnephia to be a valid taxon most closely related to Araucnephioides (sympatric in Chile). Araucnephia also shows great affinities with Lutzsimulium from Southeast Brazil and Argentina and Paracnephia from South Africa.     

Studies on the Growth of Basidiomycetes in Soil

Fructifications of basidiomycetes have been collected at LakenheathWarren, and thirty-nine species have been obtained from fivegrassland soils. The different soils have been found to havedifferent species growing upon them and most species occur onthe alkaline-slightly acid grasslands. Highly acid grasslandat Lakenheath has a poor fungus flora. Studies have been made of the regions in the soil in which themycelia of certain of these basidiomycetes occur, and attemptshave been made to isolate these fungi from soil. Some specieshave been found to inhabit the litter zone rather than the minerallayers of the soil. A few form extensive rhizomorph systemswhich may penetrate deeply into the soil. A few species of ringfungi, such as Marasmius oreades, Psalliota arvensis, and Tricholomanudum, have been found to possess a well-defined mycelial zonein the soil, from which the fungus could be isolated. Profile studies have shown that the mycelial zones of thesespecies contain a restricted population of microfungi, bothfewer species and fewer colonies, as compared with the normalsoil around. Ascomycetes, such as Arachniotus, Chaetomium, Gymnoascus,and Penicillium, have been isolated more frequently from mycelialzones than from normal soil.     

Biflavonoids in the primitive monocots Isophysis tasmanica and Xerophyta plicata

In addition to the previously characterized amentoflavone and dihydroamentoflavone, two further biflavonoids have been identified from Isophysis tasmanica: hinokiflavone and podocarpusflavone A. This is the first time these two biflavonoids have been found in the monocotyledons. Amentoflavone has also been discovered in Xerophyta plicata; this constitutes the first record of biflavonoids in Velloziaceae.     

Composition and structure of secondary fatty alcohols and ketones,isolated from Pyrus malus skin wax

A homologous series of long-chain secondary fatty alcohols from C₂₁ to C₂₉ (C₂₉ being Predominant) has been isolated from the skin wax of a Bulgarian apple variety, Bouhavitsa. The alcohols were found only in a free state. Long-chain fatty ketones, with C₂₉ again markedly prevalent, have been isolated for the first time from the skin wax of this, and another variety, Tetovka. The C₂₉-ketone from both samples was identified by means of MS as nonacosan-10-one. A complete similarity has been found in the relative amounts of C₂₉ in the mixtures of secondary alcohols, ketones and paraffins.     

Mapping of the Rf-3 nuclear fertility-restoring gene for WA cytoplasmic male sterility in rice using RAPD and RFLP markers

The cytoplasmic male sterility (CMS) of wild-abortive (WA) cytoplasm has been widely used for breeding hybrid rice. Two restorer genes for the CMS have been found by traditional genetic analysis. To tag the restorer genes we used a set of near-isogenic lines (NILs) of Zhenshan 97 carrying different genotypes for fertility restoration from IR24, to perform RAPD analysis. From the survey of 720 random primers, six RAPD markers were identified to be associated with Rf-3. Three of these OPK05-800, OPU10-1100 and OPW01-350, were mapped on chromosome 1. Two populations from the crosses between Zhenshan 97 A and a near-isogenic restorer line ZSR21 and between Zhenshan 97 A and IR24 were used for mapping Rf-3. The three RAPD markers and three RFLP markers, RG532, RG140 and RG458, were found to be closely linked to Rf-3 in the two populations. The same location of Rf-3 was also found in a population from the cross of IR58025 A//IR36/IR58025 B. At the RG532 locus, different alleles were found between two CMS lines, Zhenshan 97 A and IR58025 A, and between two restorer lines, IR24 and IR36. The use of these molecular markers closely linked to Rf-3 in facilitating the development of hybrid rice is discussed. Received: 3 January 1996 / Accepted: 17 May 1996     

A new validated HPLC-FLD method for detecting ochratoxin A in dry-cured meat and in blue cheese

In the present study, a fast and sensitive method for the quantification of ochratoxin A in two lipidicproteic food matrices has been developed. In particular, the sample preparation procedure has been optimized for dry-cured meat products and blue cheeses and tested for several validation parameters (LOD, LOQ, recovery, repeatability and within-laboratory precision). The procedure has been then applied to several dry-cured meat products and blue cheeses from the market. Ochratoxin A has been occasionally found in dry-cured and smoked ham from the market and the contamination occurred both in the outer and in the inner part of the products. Concerning the blue cheese, the occurrence of ochratoxin A is reported for the first time: OTA was occasionally found at low levels (0.1–3 μg/kg) in commercial samples of Roquefort from France and Gorgonzola from Italy, opening a new issue for risk assessment and quality control. Presented at the 29^th Mykotoxin-Workshop, Fellbach, Germany, May 14–16, 2007 Financial support: This work was partially granted by Emilia-Romagna region (projects: SIQUAL and “Safety and quality of typical pork meat production chain”)     

Aoruroides queenslandensis n. sp. (Oxyurida: Thelastomatoidea), a new nematode from Australian Panesthiinae (Blattodea: Blaberidae)

A new thelastomatid, Aoruroides queenslandensis, is described from two native Australian cockroaches, Panesthia tryoni tryoni Shaw and P. cribrata Saussure, from sub-tropical rainforest in south-eastern Queensland. Species of Aoruroides Travassos & Kloss, 1958 have previously been reported from cockroaches found in Brazil and the Philippines, but A. queenslandensis n. sp. is the first species of this genus found in Australia. The new species differs from the other members of Aoruroides principally in the position of the nerve-ring and egg morphology.     

The Isolation and Structure of Nezukone

A new tropone compound, named nezukone, has been isolated from the wood of Thuja standishii Carr, and shown to possess the structure (I). It is the first case that a tropone is found in nature.     

Evidence for horizontal transfer from streptococcus to escherichia coli of the kfid gene encoding the K5-specific UDP-glucose dehydrogenase

Capsular polysaccharides are important virulence factors both in Gram-positive and Gram-negative bacteria. A similar cluster organization of the genes involved in the synthesis of bacterial exopolysaccharides has been postulated in both cases, suggesting that these clusters evolved by module assembly. Horizontal gene transfer has been postulated to explain the polymorphism found in these cellular polymers. The cap1K and cap3A genes coding for the pneumococcal type 1 and type 3 UDP-glucose dehydrogenases, respectively, have been compared with other UDP-sugar dehydrogenases. We have observed that the evolutionary distance between Cap1K and Cap3A is approximately equal to that found between Cap1K (or Cap3A) and other UDP-GlcDH of families evolutionarily distant like KfiD, the dehydrogenase from Escherichia coli K5. On the basis of comparisons of G + C content, patterns of synonymous and nonsynonymous substitutions, dinucleotide frequencies, and codon usage bias, we conclude that the kfiD gene has been introduced into E. coli from an exogenous source, probably from a streptococcal species. Received: 26 May 1997 / Accepted: 30 July 1997     

The Widespread Occurrence of Polyphosphate in Lower Plants

The widespread occurrence of polyphosphate (PoP) in lower plantsis described. PoP granules have been cytochemically detectedin algae, slime moulds, and mosses. A systematic survey is givenof lower plants in which the occurrence of PoP has been demonstratedwith both the present studies and previous investigations. The localization of PoP in cells has been studied. PoP granuleshave been observed to be present in the cytoplasm, the cytoplasmicvacuole, and in chloro-plasts of different algae. In Zygnemataceaeand in mosses PoP granules are localized in the chloroplastswhich also contain pentose nucleic acid. A relationship between PoP, pentose nucleic acid (PNA), andgrowth has been found and is discussed. A possible in situ synthesisof PoP in chloroplasts from phosphorylated intermediates ofthe photosynthetic process is suggested.     

Isolation and physical characterization of plasmid pCCl from Corynebacterium callunae and construction of hybrid derivatives

Summary The corynebacteria seem to be the most suitable microorganisms for cloning genes involved in the production of amino acids, nucleotides, and other products of industrial interest. A plasmid, pCCl, from Corynebacterium callunae has been found with a size of 4.3 kb. A physical map obtained with restriction endonucleases is presented. pCCl has single restriction sites for Kpn I, Sma I, Bal I, and Hind III. Copy number of this plasmid has been estimated to be about 30. A number of hybrid plasmids have been constructed between pCCl and pBR329 from Escherichia coli and transformed into corynebacteria. The thiostrepton resistance gene (tsr) from Streptomyces azureus has been inserted into them.