Bi-allelic IARS mutations in a child with intra-uterine growth retardation, neonatal cholestasis, and mild developmental delay.

Recently, bi-allelic mutations in cytosolic isoleucyl-tRNA synthetase (IARS) have been described in three individuals with growth delay, hepatic dysfunction, and neurodevelopmental disabilities. Here we report an additional subject with this condition identified by whole-exome sequencing. Our findings support the association between this disorder and neonatal cholestasis with distinct liver pathology. Furthermore, we provide functional data on two novel missense substitutions and expand the phenotype to include mild developmental delay, skin hyper-elasticity, and hypervitaminosis D.

Extensive heterosis in growth of yeast hybrids is explained by a combination of genetic models.

Heterosis, also known as hybrid vigor, is the superior performance of a heterozygous hybrid relative to its homozygous parents. Despite the scientific curiosity of this phenotypic phenomenon and its significance for food production in agriculture, its genetic basis is insufficiently understood. Studying heterosis in yeast can potentially yield insights into its genetic basis, can allow one to test the different hypotheses that have been proposed to explain the phenomenon and allows better understanding of how to take advantage of this phenomenon to enhance food production. We therefore crossed 16 parental yeast strains to form 120 yeast hybrids, and measured their growth rates under five environmental conditions. A considerable amount of dominant genetic variation was found in growth performance, and heterosis was measured in 35% of the hybrid-condition combinations. Despite previous reports of correlations between heterosis and measures of sequence divergence between parents, we detected no such relationship. We used several analyses to examine which genetic model might explain heterosis. We found that dominance complementation of recessive alleles, overdominant interactions within loci and epistatic interactions among loci each contribute to heterosis. We concluded that in yeast heterosis is a complex phenotype created by the combined contribution of different genetic interactions.

Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox.

High molecular weight homologues of gp91phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for "dual oxidase" because they have both a peroxidase homology domain and a gp91phox domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. elegans. RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed cross-linking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix.

Reactions of aromatic and sulfur amino acids in ribonuclease with hydrogen atoms in water solution.

Neutral hydrogen atoms are simple, one-electron-equivalent reagents and their reactions are of fundamental interest for chemical kinetics. Functional correlation between aromatic and divalent-sulfur amino acids in ribonuclease is shown in the reaction of ribonuclease with hydrogen atoms in aqueous solution. Products of the reaction are partially characterized.

Biologicl activity and synthesis of an encephalitogenic determinant.

A 45-residue fragment of the basic protein of myelin is encephalitogenic in the rabbit and monkey but relatively inactive in the guinea pig. Synthetic peptides containing the sequence of a tryptic peptide of the fragment Thr-Thr-His-Tyr-Gly-Ser-Leu-Pro-Gln-Lys are moderately encephalitogenic.

Encephalitogenic protein: structure.

Amino acid sequences of encephalitogenic proteins from bovine cord and rabbit brain are reported. The bovine protein contains 45 residues. The rabbit protein is identical except for two isopolar substitutions, a dipeptide and amino acid deletion. Analysis of this protein and a 140-residue myelin basic protein indicates that the smaller protein is a portion of the larger encephalitogen. The larger myelin protein contains at least two encephalitogenic sites.

Experimental allergic encephalomyelitis in the rat: response to encephalitogenic proteins and peptides.

Lewis rats were used to determine the encephalitogenic activity of myelin basic protein of different species and of 45-residue fragments of basic protein. Basic protein from guinea pigs was more active than that from rats, and the fragments from the two species showed the same order of activity. Bovine basic protein was the least active of the intact proteins, and the respective fragment was inactive. Studies of serum-binding capacity did not support the hypothesis that blocking antibody played a role in this biological variation, whereas consideration of the amino acid sequences of the three fragments suggested that differences in primary structure, operating either at the sensitization or the effector phase of the immune response, could account for the variation.

Natural history of alcoholic hepatitis. IV. Glycosaminoglycuronans and collagen in the hepatic connective tissue.

The extractable and nonextractable collagen and glycosaminoglycuronans (GAG) were estimated and characterized in 32 dried, defatted human livers obtained at necropsy. 10 had normal livers. 22 of the 32 livers were from patients who drank in excess: 5 had fatty livers, 7 had alcholic hepatitis, and 10 had cirrhosis. Livers with alcoholic hepatitis or cirrhosis had significantly increased total and 1 N NaCl-extractable collagen. Only alcoholic hepatitis livers had significantly increased Tris-buffer-extractable GAG, but the amino acid composition of these GAG (proteoglycans) was no different from that of normal livers. The major fraction of these GAG had isoelectric pH (pI)

Cloning and expression of the genes involved in the production of and immunity against the bacteriocin lacticin RM.

The production of lacticin RM, a novel bacteriocin produced by Lactococcus lactis subsp. lactis EZ26, is associated with the presence of a 6-kb plasmid, pHU1. The information necessary for lacticin RM production and immunity was localized to a 2.5-kb SalI-Eco47III fragment. Sequencing analysis of this fragment revealed the presence of six open reading frames (ORFs). Deletion and mutation analyses showed that orfX and orfY are not required for lacticin RM production or immunity, whereas the other ORFs (lacA, lacF, lacG and lacI) are necessary for the bacteriocin's production. Transcription analysis indicated that lacA, lacF and lacG are organized in an operon. lacA is probably the lacticin RM structural gene. It putatively encodes a 134-amino acid peptide, and it does not share homology with known bacteriocins. The deduced LacG protein is hydrophobic and consists of six potential trans-membrane helices. lacF encodes a conserved ATP-binding domain homologous to ABC transporters known in bacteriocin immunity systems. LacF and LacG may form an active ABC transporter. Gene-disruption mutations indicated that both are required for immunity against lacticin RM. lacI encodes a small cationic protein, which is required for the production of and immunity to lacticin RM. Protection was obtained only when lacF, lacG and lacI were present together.

Reversible Inhibition of Tomato Fruit Gene Expression at High Temperature (Effects on Tomato Fruit Ripening).

The reversible inhibition of three ripening-related processes by high-temperature treatment (38[deg]C) was examined in tomato (Lycopersicon esculentum L. cv Daniella) fruit. Ethylene production, color development, and softening were inhibited during heating and recovered afterward, whether recovery took place at 20[deg]C or fruit were first held at chilling temperature (2[deg]C) after heating and then placed at 20[deg]C. Ethylene production and color development proceeded normally in heated fruit after 14 d of chilling, whereas the unheated fruit had delayed ethylene production and uneven color development. Levels of mRNA for 1-aminocyclopropane-1-carboxylic acid oxidase, phytoene synthase, and polygalacturonase decreased dramatically during the heat treatment but recovered afterward, whereas the mRNA for HSP17 increased during the high-temperature treatment and then decreased when fruit were removed from heat. As monitored by western blots, the HSP17 protein disappeared from fruit tissue after 3 d at 20[deg]C but remained when fruit were held at 2[deg]C. The persistence of heat-shock proteins at low temperature may be relevant to the protection against chilling injury provided by the heat treatment. Protein levels of 1-aminocyclopropane-1-carboxylic acid oxidase and polygalacturonase also did not closely follow the changes in their respective mRNAs. This implied both differences in relative stability and turnover rates of mRNA compared to protein and nontranslation of the message that accumulated in low temperature. The results suggest that high temperature inhibits ripening by inhibiting the accumulation of ripening-related mRNAs. Ripening processes that depend on continuous protein synthesis including ethylene production, lycopene accumulation, and cell-wall dissolution are thereby diminished.

Isolation characterization and idiotype of Lewis rat antibodies against peptide 68-88 of guinea pig myelin basic protein.

Antibodies against the 19 amino acid encephalitogenic peptide )residues 68-88) of guinea pig myelin basic protein (GPBP) were raised in Lewis (Le) rats. Anti-peptide antibodies were isolated from immune ascitic fluids by affinity chromatography using peptide 43-88-Sepharose 4B. The purified antibodies were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focusing. Immunoglobulin class was determined by radioimmunoassay. Anti-idiotypic (anti-ID) antibodies were raised in a rabbit using purified anti-peptide antibodies from a single rat. The results of these experiments showed antibody heterogeneity both within an individual anti-peptide antiserum and between antisera from different rats. Antibody activity was found in IgG1, IgG2, and IgE immunoglobulin classes. Isoelectric focusing revealed multiple bands within a population of purified antibodies with significant pattern variation from one antiserum to another. Idiotypic characterization showed various levels of cross-reactive idiotypes present in some sera while these were absent in others.

Molecular analysis and overexpression of the gene encoding endothiapepsin, an aspartic protease from Cryphonectria parasitica.

The gene, epn-1, encoding endothiapepsin (Epn), an aspartic protease (AspP) synthesized and secreted by the ascomycete fungus responsible for chestnut blight, Cryphonectria (Endothia) parasitica, was identified and characterized. Inspection of the nucleotide and deduced amino acid (aa) sequences revealed perfect agreement with the experimentally derived 330-aa sequence of mature Epn [Barkholt, Eur. J. Biochem. 167 (1987) 327-338] and an additional 89 aa of putative preprosequence. Of the nine fungal AspP characterized to date, Epn was found to be most closely related to aspergillopepsin and penicillopepsin (52% and 55% identity, respectively), proteases produced by the ascomycetes Aspergillus awamori and Penicillium janthinellum, and least related to proteases produced by the yeasts Candida albicans and Saccharomyces cerevisiae (27% and 26% identity, respectively). Epn production was found to be the same in isogenic virus-free and virus-containing strains, indicating that this AspP is not down-regulated by the presence of a hypovirulence-associated viral double-stranded RNA, as has been reported for several other secreted C. parasitica gene products. Strains containing multiple copies of epn-1 were obtained by transformation with a plasmid vector containing the cloned epn-1. One of these strains was shown to produce seven to ten times more Epn than the parental wild-type strain.

Efficacy of lamivudine for the treatment of hepatitis B virus infection after liver transplantation in children.

BACKGROUND: There is at present very little information about hepatitis B virus (HBV) infection in children after liver transplantation. This is the first study to assess the safety and efficacy of lamivudine in this patient population. METHODS: We describe three children aged 5-14 years who underwent liver transplantation for fulminant hepatitis A, hyperoxaluria, and cystic fibrosis. Despite adequate immunoprophylaxis, two of the children who were serum hepatitis B surface antigen-positive before transplantation (HBV DNA-negative by hybridization) had a reactivation of the disease, and one had a de novo HBV infection, at 12-18 months after transplantation. Lamivudine 3 mg/kg was administered on a compassionate-use basis for 14-36 months. RESULTS: After 1 month of therapy, HBV DNA disappeared from the serum in all patients by hybridization and in two patients by polymerase chain reaction. In all three children, alanine transaminase levels normalized. One child developed lamivudine resistance after 22 months with no evidence of hepatic decompensation. Repeated liver histological studies revealed progression of hepatic fibrosis in one child. All children remained serum hepatitis B surface antigen- and hepatitis B e antigen-positive. No adverse effects of the drug were noted. CONCLUSION: Lamivudine is beneficial and well tolerated in children with HBV infection after liver transplantation.

Large-scale preparation and characterization of recombinant ovine placental lactogen.

To clone ovine placental lactogen (oPL) cDNA, total RNA from sheep placental cotyledon was reverse transcribed and the single-stranded cDNA was PCR-amplified with 5' and 3' primers containing, respectively, NcoI and PstI sites. The oPL cDNA fragment amplified between these two primers extended from A(-1) to the natural stop codon. The PCR product was gel-purified and subcloned into a Puc vector and the insert was sequenced on both strands, revealing several differences relative to the published sequence: S19N, S69N, D129E and R165Q. We assume that these differences can be accounted for by the high level of individual polymorphism, which has been described in detail for PLs of different species. The insert was subcloned into NcoI/ PstI-digested pTrc99A procaryotic expression plasmid and protein expression was induced by isopropyl-1-thio-beta-D-galactopyranoside. Because of low expression, oPL's cDNA was further subcloned into pET8 procaryotic expression plasmid. Its expression in E. coli strain BL21 transformed with this vector yielded 30-40 mg/l. The expressed protein, found in the inclusion bodies, was refolded into a monomer and purified on a Q-Sepharose column to homogeneity. Structural analysis using circular dichroism revealed a spectrum similar to that of human GH (hGH) thereby indicating proper refolding. Gel filtration and binding experiments, including real-time kinetic measurements using the surface plasmon resonance method revealed that oPL forms transient homodimeric complexes with extracellular domains of prolactin receptors from rabbit, rat and bovine and with hGH receptor. The purified oPL was biologically active in an Nb2-11C cell proliferation bioassay, in its ability to stimulate beta-casein synthesis in explants of ovine and rabbit mammary gland and fat synthesis in explants of bovine mammary gland, and in a proliferation assay using FDC-P1 cells transfected with rabbit or hGH receptors.

Molecular characterization of the replicon of the Pediococcus pentosaceus 43200 pediocin A plasmid pMD136.

The pediocin A-encoding plasmid of Pediococcus pentosaceus 43200, pMD136, was characterized by restriction enzyme analysis. Analysis of its replicon was facilitated by the construction of a probe vector consisting of the Escherichia coli plasmid pSP72 and the cat gene from Staphylococcus aureus plasmid pC194. The replication region of pMD136 was localized on a 1.6-kb EcoRI/BglII fragment. Sequencing analysis revealed a non-coding region, repA, spanning the first 440 bp, followed by an open reading frame, repB, encoding a putative protein of 390 amino acids. The non-coding region contained two sets of 6-bp and two sets of 22-bp direct repeats and two sets of inverted repeats upstream of the open reading frame. Strong homology of the isolated replicon was found to theta-type replicons of Lactococcus lactis plasmids. Segregational stability assay suggested at least two regions as potentially involved in the stabilization of pMD136. The plasmid's strong homology to other theta-type replicons and its relatively high stability suggest that pMD136 belongs to the widespread family of theta-replication plasmids.

Possible synergistic effect of nisin and propionic acid on the growth of the mycotoxigenic fungi Aspergillus parasiticus, Aspergillus ochraceus, and Fusarium moniliforme.

The effects of nisin and propionic acid (PA) on aflatoxin production and on mycelial growth and spore germination of the mycotoxigenic fungi Aspergillus parasiticus, A. ochraceus, and Fusarium moniliforme were investigated. The growth of A. ochraceus was completely inhibited on media containing PA with nisin in concentrations of 0.05% PA with 1,000 ppm nisin, and 0.1% PA with 500 or 1,000 ppm nisin. The growth of both F. moniliforme and A. parasiticus was completely inhibited by PA with nisin at a concentration of 0.1% PA with 1,000 ppm nisin. Nisin alone caused a significant increase in mycelial growth when applied to A. ochraceus at 500 or 1,000 ppm and when applied to A. parasiticus at 1,000 ppm. Spore germination of A. ochraceus was completely inhibited on media containing 0.1% PA with 500 or 1,000 ppm nisin. Spores of F. moniliforme failed to germinate in 0.05% PA with 500 or 1,000 ppm nisin, whereas spores of A. parasiticus did not germinate on media containing 0.1% PA with 1,000 ppm nisin. For all three fungi tested, the inhibitory effect on mycelial growth was found to be fungistatic rather than fungicidal. The combined treatment of PA with nisin produced better fungistatic activity than treatment involving either material alone. Nisin, applied alone, did not stimulate aflatoxin production (expressed by microg toxin/mg mycelium), but the combined treatment at certain concentrations was inhibitory to aflatoxin B1 or G1. The production of aflatoxin G1, but not of B1, was stimulated in 0.05% PA with 1,000 ppm nisin and on media containing 0.1% PA with 100 ppm nisin. Nisin is currently applied in foods to prevent spoilage induced by bacteria but not by mold. The results of the present study indicate that a combined treatment of nisin in small concentrations of PA might be useful in preventing mold damage in certain foods and stored grain.

More about polyhedra-interpretation through constructions in the image plane.

This paper deals with the interpretation and feasibility check of line drawings representing polyhedral scenes. The polyhedra are of general types and there are no restrictions on camera position. The geometric consistency check and the line labeling are carried out through constructions in the image plane. An algorithm for the geometric construction is suggested, and the necessary conditions for these constructions are discussed. The image plane construction can be used for preparing labeled junction catalogs for junctions other than trihedral. In addition the paper analyzes the relation between the image plane construction and the gradient space construction suggested by Mackworth [7] for the same purpose.

A Note on Sugihara's Claim.

This correspondence debates a theorem formulated by K. Sugihara in a recent publication. The theorem deals with the mathematical structures of line drawings, and serves as a basis for a substantial part of the paper. This note claims that the theorem as formulated is not valid. The claim is substantiated by a counterexample.

The use of objects' faces in interpreting line drawings.

The two parts, which this paper is composed of, deal each with scene interpretation via gaining understanding of the faces of the objects in the scene. The first part extends the set of rules defined in a previous work regarding the assembling of all lines belonging to the same face. The set of rules, originally defined for curved object, can be extended if we confine ourselves to polyhedra. In the second part, a new concept is defined and developed, which leads to a new way of looking at polyhedral line drawings. It puts under the same roof almost all consistency checks known for polyhedra, in a natural and simple way. Geometric inconsistencies as well as interpretations inconsistencies are treated uniformly and in a straightforward manner. Through this concept a way is suggested for acquiring some understanding of back faces, and for suggesting plausible interpretation for them. The generality of this concept is demonstrated through the fact that previously known catalogs of labeled junctions can be derived directly from this concept.

Synthesis and phosphorylation of osteopontin by avian epiphyseal growth-plate chondrocytes as affected by differentiation.

The regulation of synthesis and phosphorylation of osteopontin in relation to avian epiphyseal growth-plate chondrocyte differentiation was studied in situ and in culture. Osteopontin gene expression was evaluated in the tibia growth-plate of 3-week-old chickens by in situ hybridization. The gene was expressed mainly at the lower hypertrophic zone where cartilage matrix is calcified and endochondral bone formation is initiated. Within the hypertrophic region, a poorly labeled area separated the layer of osteopontin-positive hypertrophic chondrocytes from those associated with endochondral bone formation. In culture, proliferative chondrocytes show no alkaline phosphatase activity in contrast to ascorbic acid-treated chondrocytes which display the enzyme activity. Chondrocytes not treated with ascorbic acid, exhibited lower levels of osteopontin mRNA than the treated cells. The phorbol ester TPA--an activator of protein kinase C--and to a lesser extent FGF but not EGF, stimulated osteopontin gene expression. Chondrocytes secreted low levels of phosphorylated osteopontin to the medium. EGF treatment resulted in the appearance of phosphorylated osteopontin in the medium, without affecting the synthesis of other proteins. FGF and TGF beta, but not IGF-I or IGF-II, also caused phosphorylation of osteopontin. Ascorbic acid-treated chondrocytes secreted higher levels of phosphorylated osteopontin than the non-treated cells, but addition of FGF or TPA did not stimulate osteopontin phosphorylation any further. Parathyroid hormone caused a dose-dependent attenuation of osteopontin phosphorylation and inhibited the EGF-dependent osteopontin phosphorylation. The results suggest that osteopontin gene expression and phosphorylation in chondrocytes are regulated by separate mechanisms. The response to the various controlling agents varies with the state of differentiation. Both processes--the synthesis and phosphorylation of osteopontin--are under the control of local growth factors which are involved in bone growth and calcification.