Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

A proton nuclear magnetic resonance assignment and secondary structure determination of recombinant human thioredoxin

Two-dimensional 1H NMR spectroscopy has been applied to a structural analysis of the reduced form of a recombinant human thioredoxin, a ubiquitous dithiol oxidoreductase recently isolated from an immunocompetent lymphoblastoid cell line. The sequential assignment of the spectrum, including all proline residues, has been accomplished by using experiments to demonstrate through-bond and through-space connectivities. The secondary structure has been determined by a qualitative interpretation of nuclear Overhauser effects, NH exchange data, and 3JHN alpha coupling constants. The secondary structure was found to be similar to that of the X-ray structure of Escherichia coli thioredoxin, consisting of a mixed five-stranded beta-sheet surrounded by four alpha-helices. The assignment and structural characterization of human thioredoxin was facilitated by the increased resolution and sensitivity afforded by a magnetic field strength of 600 MHz and required the use of two temperatures and two pH conditions to resolve ambiguities caused by a duplication of resonances. This duplication, extending from Phe-41 to Val-59, and including Lys-3-Ile-5, Val-24, Val-25, Asn-39, and Ile-101-Glu-103, appears to be due to heterogeneity arising from the presence or absence of the N-terminal methionine.     

Determination of the three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 phi backbone and 21 chi 1 side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.12 A for the backbone atoms and 0.90 +/- 0.17 A for all atoms. The core of the protein is formed by a triple-stranded antiparallel beta-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel beta-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel beta-sheet are connected by a long exposed loop (residues 17-30). A number of side-chain interactions are discussed in light of the structure.     

A proton nuclear magnetic resonance study of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: sequential and stereospecific resonance assignment and secondary structure

The sequential resonance assignment of the 1H NMR spectrum of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata is presented. This is carried out with two-dimensional NMR techniques to identify through-bond and through-space (less than 5 A) connectivities. Added spectral complexity arises from the fact that the sample is an approximately 1:1 mixture of two BDS-I isoproteins, (Leu-18)-BDS-I and (Phe-18)-BDS-I. Complete assignments, however, are obtained, largely due to the increased resolution and sensitivity afforded at 600 MHz. In addition, the stereospecific assignment of a large number of beta-methylene protons is achieved from an analysis of the pattern of 3J alpha beta coupling constants and the relative magnitudes of intraresidue NOEs involving the NH, C alpha H, and C beta H protons. Regular secondary structure elements are deduced from a qualitative interpretation of the nuclear Overhauser enhancement, 3JHN alpha coupling constant, and amide NH exchange data. A triple-stranded antiparallel beta-sheet is found to be related to that found in partially homologous sea anemone polypeptide toxins.     

Characterization of two divergently transcribed Dictyostelium gene pairs and identification of G-rich sequence element lying between them with the characteristics of a basal promoter element

The cysteine proteinase 1 (CP1) and cysteine proteinase 2 (CP2) genes of Dictyostelium discoideum encode coordinately expressed mRNA sequences that are inducible by extracellular cAMP. Both genes form part of divergently transcribed gene pairs. The gene proximal to CP1 is coordinately regulated and encodes a protein containing several potential zinc binding domains of the kind found in DNA binding proteins. The gene proximal to CP2 is a constitutively transcribed gene of unknown function. There are multiple, short, G-rich sequence elements between both gene pairs, and deletion of the pair of elements 200 nucleotides upstream from the CP2 gene abolishes cAMP-inducibility. A synthetic oligonucleotide, containing two copies of the G-rich element from the CP1 gene, will reconstitute cAMP-inducibility in the deletion mutant of the CP2 gene. This shows that the elements in the two genes are functionally homologous. Efficient induction requires at least two copies of the CP1 element, but their relative orientation is unimportant. Two copies in an inverted orientation are, however, inactive when moved upstream of their normal position and are incapable of conferring cAMP-inducibility on a heterologous gene. These observations suggest that these sequences are either essential promoter elements, not themselves interacting with the inducer, or that their interaction with a separate class of control sequences is necessary for inducible expression.     

Attachment and long term survival of adult rat hepatocytes in primary monolayer cultures: Comparison of different substrata and tissue culture media formulations

Summary Long-term monolayer cultures of adult rat hepatocytes were tested for their ability to glucuronize phenol red and to maintain initial levels of cell proteins, glucose consumption, and lactic acid production. Lactate dehydrogenase leakage served as an index of culture status because a high value indicates cell death. Three tissue culture (TC) media formulations were the main variables introduced to determine ideal conditions for cell survival in vitro. Investigations of long-term cultures were preceded by studies of hepatocyte attachment to polystyrene surfaces. This attachment was influenced by the amount of substrate deposited and the number of cells seeded, but not by the uniformity of the substrate coating. A statistical analysis of our data revealed that in the absence of fetal bovine serum (FBS), air dried collagen (ADC) and Biomatrix (BMX) were superior to saline precipitated collagen and fibronectin as attachment substrates. In the presence of 10% FBS, all of the substrates performed equally. Chee's Medium (CEM) proved to be the best for preserving cell proteins over a time course of 28 d and Williams' E medium also performed adequately up to 14 d. The glucuronization of phenol red was at 50% of initial values at Day 7 in CEM-ADC hepatocytes in contrast to 30% for cells in Williams' E medium and 5% for cells grown in Waymouth's. At 14 d glucuronization was still present at 40% of original values in CEM-ADC cells but had ceased in the other two media. When BMX was used, none of the TC media supported glucuronization levels comparable to ADC cells. This research was supported in part by grant 1R01-AM-26520 from the National Institute of Arthritis, Diabetes and Digestive Kidney Diseases, NIH, Bethesda, Maryland.     

Locus determining the human sperm-specific lactate dehydrogenase,LDHC, is syntenic with LDHC

From the data presented in this report, the human LDHC gene locus is assigned to chromosome 11. Three genes determine lactate dehydrogenase (LDH) in man. LDHA and LDHB are expressed in most somatic tissues, while expression of LDHC is confined to the germinal epithelium of the testes. A human LDHC cDNA clone was used as a probe to analyze genomic DNA from rodent/human somatic cell hybrids. The pattern of bands with LDHC hybridization is easily distinguished from the pattern detected by LDHA hybridization, and the LDHC probe is specific for testis mRNA. The structural gene LDHA has been previously assigned to human chromosome 11, while LDHB maps to chromosome 12. Studies of pigeon LDH have shown tight linkage between LDHB and LDHC leading to the expectation that these genes would be syntenic in man. However, the data presented in this paper show conclusively that LDHC is syntenic with LDHA on human chromosome 11. The terminology for LDH genes LDHA, LDHB, and LDHC is equivalent to Ldhl, Ldh2, and Ldh3, respectively.     

Acidification and alkalinization of soils

Acidification or alkalinization of soils occurs through H⁺ transfer processes involving vegetation, soil solution and soil minerals. A permanent change in the acid neutralizing capacity of the inorganic soil fraction (ANC_(s)),i.e. soil acidification (ΔANC<0) or soil alkalinization (ΔANC>0), results from an irreversible H⁺ flux. This irreversible H⁺ flux can be caused either by direct proton addition or depletion, by different mobility of components of the ANC_(s) or by a permanent change in redox conditions. The contributions of (a) acidic atmospheric deposition, (b) nitrogen transformations, (c) deprotonation of CO₂ and of organic acids and protonation of their conjugate bases, (d) assimilation of cations and anions by the vegetation, (e) weathering or reverse weathering of minerals and (f) stream output to changes in the ANC_(s) are illustrated by means of H⁺ budgets for actual soils and watersheds.