1H NMR studies of the mercuric ion binding protein MerP: Sequential assignment,secondary structure and global fold of oxidized MerP

Summary The oxidized form of the mercuric ion binding protein MerP has been studied by two-dimensional NMR. MerP, which is a periplasmic water-soluble protein with 72 amino acids, is involved in the detoxification of mercuric ions in bacteria with resistance against mercury. The mercuric ions in the periplasmic space are first scavenged by the MerP protein, then transported into the cytoplasm by the membrane-bound transport protein MerT, and finally reduced to elementary (nontoxic) mercury by the enzyme mercuric reductase. In this work, the ¹H NMR spectrum of oxidized MerP (closed disulfide bridge) has been assigned by using homonuclear 2D NMR techniques. The secondary structure and global fold have been inferred from the nuclear Overhauser effect (NOE) data. The secondary structure comprises four -strands and two -helices, in the order ₁₁₂₃₂₄. The protein folds into an antiparallel -sheet, ₂₃₁₄, with the two antiparallel helices on one side of the sheet. The folding topology is similar to that of acylphosphatase, the activation domain of porcine pancreatic procarboxypeptidase B, the DNA-binding domain of bovine papillomavirus-1 E2 and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins. However, there is no structural similarity between MerP and other bacterial periplasmic binding proteins.     

Polymerization Sites in the D-Domain of Human Fibrin(ogen)

The present work deals with localization of previously unknown polymerization sites of the fibrin DD-fragment. D-dimer we obtained has a pronounced inhibitory effect on fibrin polymerization (IC₅₀ = 0.06 M). The inhibitory effect of the D-fragment disappeared after reduction and carboxymethylation. However, polypeptide chains _DD (B134-461) and _DD (63-411)₂ of the DD-fragment, isolated by preparative electrophoresis, displayed their inhibitory activity. For instance, the rates of fibrin protofibril lateral association were decreased twice in the presence of _DD and _DD chains at their molar ratios to fibrin of 0.40 and 0.15, respectively. The IC₅₀ values for _DD and _DD were 0.24 and 0.10 M, respectively. Highly specific inhibition of protofibril lateral association suggests that the protofibril lateral association sites are located in B134-461 and 63-411 regions of the fibrin D-domain. Our data confirm those reported by Doolittle et al. regarding the -chain and a hypothesis about -chain of fibrin D-domain (Yang, Z., Mochalkin, I., and Doolittle, R. F. (2000) Biochemistry, 97, 14156-14161).     

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.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

Diversity of the mouse T cell receptor Cγ1 gene: structural analysis in C57BL/Ka

We have isolated an unusual T cell receptor chain cDNA clone (7.1) from a library made from RNA derived from adult thymus of C57BL/Ka mice. This cDNA clone corresponds to the appropriately processed C1 constant region exons preceded by 1.5 kb of J-C1 intron. The 7.1 coding region is extremely homologous to the C1 gene of BALB/c mice, differing at the protein level by a single deletion (alanine 139) and a single substitution. This latter change eliminates the sole N-linked sugar attachment site, providing a basis for strain-specific glycosylation patterns. The J-C1 intronic region contains two DNA segments (termed J1 and J2) that are highly reminiscent of joining (J) segments; both have potentially functional recombination and donor splice sequences flanking an open reading frame. Northern analysis suggests that 7.1 may be derived from a large, variable region-containing precursor.     

Utilization and degradation of lindane by soil microorganisms

Of 147 microorganisms isolated from a loamy sand, 71 showed good growth with lindane (-1,2,3,4,5,6-hexachlorocyclohexane) and produced chloride in an aqueous medium. Thirteen soil microorganisms were selected to study the utilization of lindane. Lindane was metabolized by the microbes to -2,3,4,5,6-pentachloro-1-cyclohexene (-PCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), -3,4,5,6-tetrachloro-1-cyclohexene (-TCCH), and pentachlorobenzene (PCB). Cells of Pseudomonas sp. No. 62 grown on lindane simultaneously adapted to -PCCH, -TCCH, -TCCH, -TCCH, PCB, 1,2,3,4-tetrachlorobenzene (1,2,3,4-TCB) and 1,2,4,5-tetrachlorobenzene (1,2,4,5-TCB). The bacteria degraded each of these chemicals at least partially as indicated by an increased rate of oxygen consumption.Abbreviations Lindane -1,2,3,4,5,6-hexachlorocyclohexane - -PCCH -2,3,4,5,6-pentachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - -TCCH -3,4,5,6-tetrachloro-1-cyclohexene - PCB pentachlorobenzene - 1,2,3,4-TCB 1,2,3,4-tetrachlorobenzene - 1,2,3,5-TCB 1,2,3,5-tetrachlorobenzene - 1,2,4,5-TCB 1,2,4,5-tetrachlorobenzene - 1,2,3-tCB 1,2,3-trichlorobenzene - 1,2,4-tCB 1,2,4-trichlorobenzene - 1,3,5-tCB 1,3,5-trichlorobenzene - 1,2-DCB 1,2-dichlorobenzene - 1,3-DCB 1,3-dichlorobenzene - 1,4-DCB 1,4-dichlorobenzene - MCB monochlorobenzene Contribution No. 631, Research Institute, Agriculture Canada, University Sub Post Office, London, Ontario N6A 5B7     

Rapid protein fold determination using secondary chemical shifts and cross-hydrogen bond 15N-13C′ scalar couplings (3hbJNC′)

The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small / protein chymotrypsin inhibitor 2. Dihedral angle restraints for the and angles of 32 out of 64 residues could be obtained from secondary chemical shift analysis with the TALOS program (Corneliscu et al., 1999a). This information was supplemented by 18 hydrogen-bond restraints derived from experimentally measured cross-hydrogen bond ^3hbJ_NC coupling constants. These experimental data were sufficient to generate structures that are as close as 1.0 Å backbone rmsd from the crystal structure. The fold is, however, not uniquely defined and several solutions are generated that cannot be distinguished on the basis of violations or energetic considerations. Correct folds could be identified by combining clustering methods with knowledge-based potentials derived from structural databases.     

Conformation of the circular dumbbell d〈pCGC-TT-GCG-TT〉: Structure determination and molecular dynamics

Summary The circular DNA decamer 5-dpCGC-TT-GCG-TT-3 was studied in solution by means of NMR spectroscopy and molecular dynamics in H₂O. At a temperature of 269 K, a 50/50 mixture of two dumbbell structures (denoted L2L2 and L2L4) is present. The L2L2 form contains three Watson-Crick C-G base pairs and two two-residue loops in opposite parts of the molecule. On raising the temperature from 269 K to 314 K, the L2L4 conformer becomes increasingly dominant (95% at 314 K). This conformer has a partially disrupted G(anti)-C(syn) closing base pair in the 5-GTTC-3 loop with only one remaining (solvent-accessible) hydrogen bond between NH of the cytosine dC(1) and O6 of the guanine dG(8). The opposite 5-CTTG-3 loop remains stable. The two conformers occur in slow equilibrium (rate constant 2–20 s^–1). Structure determination of the L2L2 and L2L4 forms was performed with the aid of a full relaxation matrix approach (IRMA) in combination with restrained MD. Torsional information was obtained from coupling constants. Coupling constant analysis (³J_HH, ³J_HP, ³J_CP) gave detailed information about the local geometry around backbone torsion angles , , and , revealing a relatively high flexibility of the 5-GTTC-3 loop. The values of the coupling constants are virtually temperature-independent. Weakly constrained molecular dynamics in solvent was used to sample the conformational space of the dumbbell. The relaxation matrices from the MD simulation were averaged over r^–3 to predict dynamic NOE volumes. In order to account for the 1:1 conformational mixture of L2L2 and L2L4 present at 271 K, we also included S² factors and r^–6 averaging of the r^–3-averaged relaxation matrices. On matrix averaging, the agreement of NOE volumes with experiment improved significantly for protons located in the thermodynamically less stable 5-GTTC-3 loop. The difference in stability of the 5-CTTG-3 and 5-GTTC-3 loops is mainly caused by differences in the number of potential hydrogen bonds in the minor groove and differences in stacking overlap of the base pairs closing the minihairpin loops. The syn conformation for dC(1), favored at high temperature, is stabilized by solvation in the major groove. However, the conformational properties of the dC(1) base, as deduced from R-factor analysis and MD simulations, include a large flexibility about torsion angle .     

A cyto-biochemical study of the ‘canal’ formation in the yeast cell wall

Summary A distinction between the chemical composition of ultrastructurally modified regions and the rest of the cell wall (canals) of the yeast Schwanniomyces occidentalis was shown by cytochemical staining of cell wall polysaccharides. The formation of canals was induced by cultivation of yeasts on hydrocarbons and was parallelled by the enhancement of -glucosidase, -glucanase and -mannosidase activities which were all capable of degrading cell wall polysaccharides. The presence of cycloheximide prevented canal formation. We assume that these hydrolases modified definite cell wall regions transforming them into canals.     

The structure of the incompatibility factors of Schizophyllum commune: Constitution of the three classes of B factors

Summary The B factors of Schizophyllum commune are of 3 classes: The high recombining class I has 7 alleles and 7 alleles; the low recombining classes are class II with 7 allels and probably 2 alleles and class III with probably 2 (or also 2) alleles and 7 allels. A fourth hypothetical class (-) was not found and either does not exist or is indistinguishable from class III by the tests employed. The and alleles differ from and by either (a) mutations affecting both mating specificity and recombination frequency, or (b) deletions involving most of the B region.The research was supported by a grant from the Atomic Energy Commission of the U.S. No. (30-1)-3875 and was performed at the Biological Laboratories, Harvard University, Cambridge, Mass., U.S.A.     

Specific Cd2+ uptake of encapsulated Aureobasidium pullulans biosorbents

Calcium alginate capsules containing Aureobasidium pullulans cells were prepared by an in-situ one-step method. The Cd²⁺uptakes of free biosorbent and capsule biosorbent were described well by the Freundlich isotherms. The Cd²⁺ uptake of capsule biosorbent was lower than that of free biosorbent with Cd^{2 +}at 100 mg l^–1. The total cadmium uptake of capsule biosorbent increased with the increase in encapsulated cell density and plateaued at a value of 23 g Cd²⁺/capsule with 150 g dry biosorbent l^–1 core volume of a capsule. The specific cadmium uptake of encapsulated biosorbent was 85% to that of free biosorbent at 35 g biosorbent dry weight l^–1 core volume of a capsule, decreased to 35% at 176 g biosorbent dry weight l^–1.     

Long-term continuous reaction of immobilized β-fructofuranosidase

Summary -Fructofuranosidase was immobilized by alginate gel at high efficiency (92 %). The extreme long-term continuous reaction (half-life, 275 days) was achieved by the immobilized enzyme using sucrose at high concentration (500 mg ml^–1) to produce fructo-olicosaccharides, such as 1-kestose (Fru21Fru21aGlc) and nystose (Fru21Fru21Fru21aGlc).     

Abscisic acid as an endogenous component in lettuce fruits,Lactuca sativa L. cv. Grand Rapids. Does it control thermodormancy?

Summary Abscisic acid (ABA) has been unequivocally identified as an endogenous component of seeds of Grand Rapids lettuce. Both free and bound ABA levels were followed during imbibition at various temperatures but no clear role for ABA in the imposition of thermodormancy emerged. Furthermore it was apparent that different seed batches showed different ratios of free to bound ABA and patterns of changes. An unsuccessful attempt was made to identify bound ABA, presumed to be the glucosyl ester.Abbreviations ABA abscisic acid - ABA Me ester methyl abscisate - ECD electron capture detector - FID flame ionisation detector - GC-MS combined gas liquid chromatography, mass spectrometry - GLC gas liquid chromatography - PVP polyvinyl-pyrrolidone - TLC thin layer chromatography     

Determination of three enzyme polymorphisms in one electrophoretic step on horizontal polyacrylamide gels

Summary A method that allows separation and determination of three enzyme polymorphisms in one polyacrylamide gel is described. Common phenotypes of adenylate kinase, adenosine deaminase and phosphoglucomutase are diagnosed without difficulties. There are small migration differences between PGM isozymes e and d.

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Zusammenfassung Es wird eine Methode beschrieben, die Trennung und Darstellung von drei Enzympolymorphismen in einem Polyacrylamid-Gel ermöglicht. Die Normtypen der Adenylatkinase, Adenosindeaminase und Phosphoglucomutase sind zweifelsfrei zu differenzieren. Geringe Wanderungsunterschiede ergeben sich zwischen den Zonen e und d der PGM Isoenzyme.

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(Direktor: Prof. Dr. W. Janssen)     

The α and γ subunits of 7S nerve growth factor are present in excess concentrations in the mouse submandibular gland

Summary The 7S nerve growth factor molecule, found in the mouse submandibular gland, is comprised of three distinct protein subunits named , and -NGF. In this paper, radioimmunoassays specific for each subunit were used to measure the concentrations of these subunits in homogenates of mouse submandibular gland. It was determined that there were excess concentrations of both the and subunits, more than enough to bind all of the -NGF in the gland to form 7S-NGF. The radioimmunoassay data was confirmed by gel filtration experiments. In the gel filtration experiments, the excess and subunits eluted at positions which would indicate that these excess subunits were free and not bound in the 7S-NGF complex. The identity of the excess and subunits was substantiated by ion exchange chromatography, isoelectric focusing polyacrylamide gels and immunoblotting experiments. In conclusion, there are considerable quantities of and subunits present in the submandibular gland which are not bound to -NGE The functional significance of these excess concentrations of the and subunits is not known.     

6′-Mercaptoguanosine-resistance is related with purF gene encoding 5′-phosphoribosyl-1′-pyrophosphate amidotransferase in inosine-5′-monophosphate overproducing Brevibacterium ammoniagenes

From the gene library constructed with the chromosomal DNA of 6-mercaptoguanosine (MGS)-resistant strain Brevibacterium ammoniagenes IPR-1, a DNA fragment which conferred MGS-resistance to the wild-type strain B. ammoniagenes ATCC6872 was cloned. The purF gene encoding 5-phosphoribosyl-1-pyrophosphate amidotransferase was identified from this fragment and its nucleotide sequence was determined. Wild type purF gene was also cloned by polymerase chain reaction using chromosomal DNA of ATCC6872 as the template and its sequence was determined. Two nucleotides, 583 A and 1065 A, of MGS-resistant purF gene had been changed from 583 G and 1065 G by mutagenesis, respectively. Both changes at position 583 and 1065 were proved to be responsible for MGS-resistance by site-directed mutagenesis.     

Nucellar callus of ‘Femminello’ lemon,selected for tolerance toPhoma tracheiphila toxin,shows enhanced release of chitinase and glucanase into the culture medium

Phoma tracheiphila is the causative agent of the disease mal secco. Citrus cultivars differ substantially in respect to their sensitivity to the pathogenP. tracheiphila and its toxin. Some cultivars (e.g., Femminello lemon) are inherently sensitive while others (e.g., Tarocco orange) are tolerant. Cell lines derived from nucellar tissue of Femminello, Tarocco and a cell line selected for tolerance to the fungal toxin (Femminello-S) were used to study host-pathogen interaction. Our results showed that calli or conditioned media of Tarocco and Femminello-S inhibited the size of co-cultivated fungal colonies when compared to Femminello. In addition, conditioned medium of Tarocco as well as FemminelloS, but not Femminello, promoted bursting of hyphal tips. A ten-fold increase in chitinase and glucanase enzymatic activity, as evaluated by radiometric assay and laminarin hydrolysis respectively, was detected in Femminello-S extracellular extracts as compared to Femminello. An increase in chitinase was also shown by immunoblot analysis. Our findings suggest a positive correlation between the presence of chitinase and glucanase in the conditioned media of the cultured cells and the tolerance of those cells toP. tracheiphila toxin.     

Site inhomogeneity and exciton delocalization in the photosynthetic antenna

The influence of energy disorder on exciton states of molecular aggregates (the dimer and the circular aggregate) was analyzed. The dipole strength and inhomogeneous line shapes of exciton states were calculated by means of numerical diagonalization of Hamiltonian with diagonal energy disorder without intersite correlation. The disorder degree corresponding to destruction of coherent exciton states was estimated. The circular aggregates were treated as a model of light-harvesting antenna structures of photosynthetic bacteria. It was concluded that the site inhomogeneity typical for LH1 and LH2 complexes of purple bacteria cannot significantly influence the exciton delocalization over the whole antenna.Abbreviations BChl- bacteriochlorophyll - LH1 and LH2- core and peripheral light-harvesting complexes from purple bacteria - RC- reaction center     

Cryptomonad biliproteins: Bilin types and locations

Two crytophycean phycocyanins (Cr-PCs), Hemiselmis strain HP9001 Cr-PC 612 and Falcomonas daucoides Cr-PC 69 were purified and characterized with respect to bilin numbers, types and locations. Each biliprotein carried one bilin on the subunit and three on the subunit. Cr-PC 612 carried phycocyanobilin at -Cys-18, -Cys-82, and -Cys-158, and a doubly-linked 15,16-dihydrobiliverdin at -DiCys-50,61. Cr-PC 569 carried phycocyanobilin at -Cys-18 and -Cys-82, a singly-linked Bilin 584 at -Cys-158, and a doubly-linked Bilin 584 at -DiCys-50,61. This work, in conjunction with earlier studies on Cr-PE 545, Cr-PE 555, Cr-PE 566, and Cr-PC 645, shows that there is no conserved location for the bilin with longest wavelength visible absorption band among these proteins, and, consequently, that there is no conserved energy transfer pathway common to all native cryptophycean biliproteins. Only phycocyanobilin or phycoerythrobilin is found at -Cys-82; there is greater bilin variability at the other three attachment sites.Abbreviations Cr-PC cryptophycean phycocyanin - Cr-PE cryptophycean phycoerythrin - DBV 15,16-dihydrobiliverdin - MBV mesobiliverdin - PCB phycocyanobilin - PEB phycoerythrobilin - HPLC high performance liquid chromatography - TFA trifluoroacetic acid