A detailed serological analysis of a xenogeneic anti-Ia serum

Rabbit anti-mouse-Ia serum was raised against Ia specificities present in CBAJH (H-2 _k) serum. This xenogeneic antiserum was considered to react with similar specificities to those detected by mouse anti-Ia^k alloantisera and more evidence is now presented for this contention. By absorption, the xenogeneic antiserum was found to react with spleen, lymph node, bone marrow, and thymus, reactions similar to that found with the allogeneic anti-Ia^k antiserum. Furthermore, red cells, platelets, brain, kidney, and liver could not absorb the activity from the xenogeneic antiserum, demonstrating the selective tissue distribution of the antigens reactive with this serum. This reactive population was previously shown to consist of B cells and a subpopulation of T cells. In a backcross study of (C57BL/6 × A)F₁ × C57BL/6, the rabbit anti-Ia and mouse anti-Ia reactions were found to segregate together, and some evidence for the genetic regulation of the expression of Ia specificities was also found. By direct testing, and by absorption testing using a number of strains, the xenogeneic antiserum was shown to contain high titers of antibody to Ia.1, 3, 7, 15, and 17; lower titers to Ia.19, and 22; little antibody to Ia.18, and no reaction for the private specificity Ia. 2, although the multiple absorptions required to define these specificities may have observed some reactions. The data indicate that the xenogeneic and allogeneic anti-Ia_k antisera recognize similar Ia determinants, which map to theLA, IE andIC subregions of theH-2 complex. These have been given the same specificity designation as the allogeneic specificities, but they are separately identified by a prime (').     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Nuclear and cytoplasmic gene control of resistance to loose smut (Ustilago tritici (Pers.) Rostr.) in wheat (Triticum aestivum L.)

Summary Using disomic chromosome substitution lines based on the susceptible wheat cultivar Chinese Spring, loose smut resistance of wheat cultivars Hope and Thatcher was shown to be conferred in each case by a single dominant major gene carried on chromosome 7 A (Hope) or 7 B (Thatcher). Partial resistance was determined by genes on an additional eight Hope or seven Thatcher chromosomes, and similarities were evident between the partial resistance genotypes ofHope and Thatcher. Chinese Spring exhibited a mean infection value of approximately 50%, indicating a significant level of partial resistance, which was found to be due, in part, to genes on the homoeologous chromosome arms 1 As, 1 Es and 1 Ds, and to cytoplasmic genes. Substitution of the Chinese Spring nucleus into the cytoplasm of Aegilops squarrosa, Ae. variabilis or Ae. mutica resulted in increased susceptibility to Ustilago tritici. Several alloplasmic lines of the resistant wheat cultivars Selkirk and Chris exhibited race-specific susceptibility to U. tritici.     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

Somatic embryogenesis in Simarouba glauca

Frequency of somatic embryogenesis from callus cultures derived from immature cotyledon explants of Simarouba glauca Linn. was highest on solid MS medium supplemented with 11.1 M benzyladenine and 13.42 M -naphthaleneacetic acid. On transfer of the somatic embryos into maturation medium containing half-strength MS medium supplemented with 1.89 M abscisic acid (ABA) and 2% (w/v)sucrose, 20–25 % of embryos germinated within 20 days of culture with distinct cotyledon, hypocotyl and radicle.     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Interspecies exchange of β 2-microglobulin and associated MHC and differentiation antigens

Radiolabeled human ₂-microglobulin (₂m) can bind to mouse histocompatibility (H-2) antigens on the cell surface or to partially purified H-2 antigens in solution. The complexes containing human ₂m and H-2 antigens from C3H (H-2^k) mice could be immunoprecipitated specifically with alloantisera, rabbit anti-H-2 xenoantisera, and with monoclonal H-2-specific antibodies. Specific association with H-2 antigens was also observed with other haplotypes. The only exception was B10.D2 (H-2 ^d ) from which complexes containing human ₂M could only be precipitated with anti-H-2 xenosera. Thus radiolabeled human ₂M can be used as a specific label for mouse H-2 antigens in precipitation and radioimmunoassays. The application of this finding extends to major histocompatibility complex antigens of other species, and to differentiation antigens with primary association with ₂m.Abbreviations used in this paper MHC major histocompatibility complex - ₂m ₂-microglobulin - LcH Lens culinaris hemagglutinin     

Aldimine to ketoamine isomerization (Amadori rearrangement) potential at the individual nonenzymic glycation sites of hemoglobin a: Preferential inhibition of glycation by nucleophiles at sites of low isomerization potential

The relative roles of the two structural aspects of nonenzymic glycation sites of hemoglobin A, namely the ease with which the amino groups could form the aldimine adducts and the propensity of the microenvironments of the respective aldimines to facilitate the Amadori rearrangement, in dictating the site selectivity of nonenzymic glycation with aldotriose has been investigated. The chemical reactivity of the amino groups of hemoglobin A forin vitro reductive glycation with aldotriose is distinct from that in the nonreductive mode. The reactivity of amino groups of hemoglobin A toward reductive glycation (i.e., propensity for aldimine formation) decreases in the order Val-1(), Val-1(), Lys-66(), Lys-61(), and Lys-16(). The overall reactivity of hemoglobin A toward nonreductive glycation decreased in the order Lys-16(), Val-1(), Lys-66(), Lys-82(), Lys-61(), and Val-1(). Since the aldimine is the common intermediate for both the reductive and nonreductive modification, the differential selectivity of protein for the two modes of glycation is clearly a reflection of the propensity of the microenvironments of nonenzymic glycation sites to facilitate the isomerization reaction (i.e., Amadori rearrangement). A semiquantitative estimate of this propensity of the microenvironment of the nonenzymic glycation sites has been obtained by comparing the nonreductive (nonenzymic) and reductive modification at individual glycation sites. The microenvironment of Lys-16() is very efficient in facilitating the rearrangement and the relative efficiency decreases in the order Lys-16(), Lys-82(), Lys-66(), Lys-61(), Val-1(), and Val-1(). The propensity of the microenvironment of Lys-16() to facilitate the Amadori rearrangement of the aldimine is about three orders of magnitude higher than that of Val-1() and is about 50 times higher than that of Val-1(). The extent of nonenzymic glycation at the individual sites is modulated by various factors, such as thepH, concentration of aldotriose, and the concentration of the protein. The nucleophiles—such as tris, glycine ethyl ester, and amino guanidine—inhibit the glycation by trapping the aldotriose. The nonenzymic glycation inhibitory power of nucleophile is directly related to its propensity to form aldimine. Thus, the extent of inhibition of nonenzymic glycation at a given site by a nucleophile directly reflects the relative role ofpK _a of the site in dictating the glycation at that site. The nonenzymic glycation of an amino group of a protein is an additive/synergestic consequence of the propensity of the site to form aldimine adducts on one hand, and the propensity of its microenvironment to facilitate the isomerization of the aldimines to ketoamines on the other. The isomerization potential of microenvironment plays the dominant role in dictating the site specificity of the nonenzymic glycation of proteins.     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

MHC restriction of male-antigen-specific T helper cells collaborating in antibody responses

By priming female C57BL/6 mice with syngeneic male spleen cells and enriching inguinal and paraaortic lymph node cells in long-term culture (LTC) by repeated restimulations, H-Y-specific T helper cells can be produced. In response to male spleen cells carrying I-Ab antigens these cells activate antigenexpressing B cells to secrete polyclonal antibody. Before the end of the second week in LTC it was impossible to detect any helper activity. Induction of plaque-forming cells (PFC) also requires simultaneous recognition of antigen and I-A-encoded determinants in the stimulator-responder spleen-cell population. The testing of spleen cells fromH-2 recombinant strains as stimulator-responders to anti-H-Y helper T cells of C57BL/6 origin also revealed that other genes, telomeric toI-A, control the magnitude of both specific T-cell proliferation and helper-dependent B-cell activation.     

Gene structure of the ‘large’ sialidase isoenzyme fromClostridium perfringens A99 and its relationship with other clostridialnanH proteins

Clostridium perfringens possesses two sialidase isoenzymes of different molecular weight. Almost 90% of the gene encoding the large form was found on a 3.1 kb chromosomal fragment (Sau3AI) of strain A99 by hybridization with probes developed from the N-terminal protein sequence and from commonly conserved sialidase motifs (Asp-boxes), whereas the remaining 3-terminal part was detected on a 2.1 kb fragment (Hind III) of chromosomal DNA. After combination of both fragments, the resultingE. coli clones expressed sialidase activity, the properties of the recombinant sialidase corresponding with those of the wild type enzyme. The entire chromosomal fragment of 3665 bp encompasses the complete sialidase gene of 2082 bp corresponding to 694 amino aids, from which a molecular weight of 72956 for the mature protein can be deduced. The first 41 amino acids are mostly hydrophobic and probably represent a signal peptide. The sialidase structural gene follows a non-coding region with an inverted repeat and a ribosome-binding site. Upstream from the regulatory region, another open reading frame (ORF) was detected. The 3-terminus of the sialidase structural gene is directly followed by a further ORF of unknown function, which possibly encodes a putative permease or the acylneuraminate pyruvate-lyase involved in sialic acid catabolism. The primary structure of the large isoenzyme is very similar to the sialidase ofClostridium septicum (55% identical amino acids), whereas the homology with the small form of the same species is comparatively low (26%).     

Tryptic peptide comparison of Ia antigen α and β polypeptides from the I-A Mutant B6.C-H-2 bm12 and its congenic parental strain B6

Previous studies of the B6.C-H-2 bm12 (bm12) strain have demonstrated the presence of a mutation localized to the I-A subregion of the mouse H-2 major histocompatibility complex. This mutation has been shown to be responsible for defects in Ir-gene function and in Ia and MLR determinants. A comparison of the molecular size of the bm12 mutant and the parental B6 Ia-antigen component polypeptides failed to demonstrate any differences in the and polypeptides. Thus, no major structural additions or deletions are present in the Ia and chain polypeptide or carbohydrate structure. A significant decrease in the amount of invariant (31K) polypeptide was, however, consistently observed in the bm12 Ia antigen preparations. Tryptic peptide comparisons of ¹⁴C B6 and ³H bm 12 and polypeptides demonstrated a limited number of peptide differences in the bm12 polypeptide but none in the bm12 polypeptide. The significance of these biochemical mutations and altered biological phenomena are discussed in relation to a model of the immunological interaction sites on Ia antigens.     

Iron sulfur proteins of the purple sulfur bacterium Ectothiorhodospira shaposhnikovii

In addition to several cytochromes three iron sulfur proteins were detected in mixotrophically grown cells of Ectothiorhodospira shaposhnikovii, a member of the Chromatiaceae. They were identified as a bacterial ferredoxin and two high potential iron sulfur proteins (HIPIPs). The two HIPIPs were purified and characterized. They were named according to their differing retention times on a DEAE-cellulose column using a continuous NaCl gradient: early and late HIPIP. The HIPIPs contain 4 mol of non-heme iron and 4 mol of acid labile sulfur per mol protein. Under the conditions of purification the early HIPIP (E _{m, 7}+270 mV) was present in a semi-reduced state. Using ion-exchange chromatography the early HIPIP could be split into a reduced green-brown (pI=3.7) and an oxidized red-brown (pI=3.9) fraction. The late HIPIP (pI=3.8) showed a midpoint potential of only+155 mV, the lowest redox potential of a HIPIP described so far.Non-common abbreviations HIPIP high potential iron sulfur protein - MOPS 3(N-morpholino)propane sulfonate - SDS sodium dodecylsulfate     

Structural evidence that the small subunit found associated with the TL antigen is β 2-microglobulin

Comparative tryptic peptide mapping and partial amino-terminal primary sequence analysis of the light chain component associated with the TL antigens showed that the small subunit of TL was identical to the ₂m light chain associated with the H-2K or D product of the same strain. Peptide comparison of the ₂m from the Tla products of an A strain X-ray induced leukemia RADA1 (Tla ^a) and of a C57BL/6 strain X-ray induced leukemia ERLD (Tla ^b) showed differences to the extent of 25–35% in their peptides. This is consistent with previous results showing ₂m allelic variations between these mouse strains. The data prove the structural identity of the ₂m molecules from TL and H-2K, D antigens as well as reveal the strain specific polymorphism of the ₂m associated with these products.     

Molecular cloning and expression of multiple cellulase genes of Ruminococcus flavefaciens strain 186 in Escherichia coli

Summary Cellulase genes of the ruminant micro-organism Ruminococcus flavefaciens strain 186 have been cloned and expressed in Escherichia coli using the bacteriophage vector NM1149. Twenty-six clones showed expression of endo--1,4-d-glucanases and were divided into four groups according to their insert sizes of approximately 2, 3, 4 or 9 kilobases (kb). Two of the clones with 4 kb inserts also showed exo--1,4-d glucanase activity while two clones with 9 kb inserts showed -glucosidase activity. One of the clones with 9 kb inserts (M903) showed the activities of all three cellulase activities. In addition, two of the 4 kb-insert clones and one 9 kb-insert clone degraded Avicel (PH101).     

Yeast ribosomal proteins

Summary Homology maps between bacteriophages 81, 80 and were constructed on the basis of electron microscope observation of DNA heteroduplexes. In 81/80 heteroduplex, the left half and the right terminal region of 13% the total molecular length were highly homologous, while the remaining region covering the early gene cluster was entirely nonhomologous. In 81/ heteroduplex, high-degree homologies were detected at the left 14% terminal region covering the head gene cluster, the central 3.8% region covering the att-int-xis region and the 1.3% Q homology region. Low-degree homologies of shorter length were scattered at the tail gene cluster, b2 region, cIII region, PQ region and SR region. Comparing our results with the homology maps of other lambdoid phages reported by Simon et al. (1971) and Fiandt et al. (1971), a phylogenic relation of 81 to other lambdoid phages and the role of recombination in the course of divergence of lambdoid phages are discussed.     

The partial amino acid sequence of a murine Ia molecule

Eleven of the amino terminal 14 amino acid residues have been assigned in the chain of the murine I-C^d subregion molecule. The murine chain shows no homology with P29 (the putative human chain equivalent), the chain of guinea pig Ia. 4, 5, or the murineI-A subregion chain.     

The fuc1 gene product (20 kDa FUC1) of Pisum sativum has no α-L-fucosidase activity

An -L-fucosidase purified from pea (Pisum sativum L. cv Alaska) epicotyl was previously described as a cell wall enzyme of 20 kDa that hydrolyses terminal -L-fucosidic linkages from oligosaccharide fragments of xyloglucan. cDNA and genomic copies were further isolated and sequenced. The predicted product of the cDNA and the genomic clone (fuc1), was a 20 kDa protein containing a signal peptide and five cysteines. This was the first -L-fucosidase gene to be cloned in plants but its fucosidase activity has not been demonstrated. Here, our biochemical and immuno analyses suggest that fuc1 does not encode an -L-fucosidase. Pea fuc1 expressed in Escherichia coli, insect cells and Arabidopsis thaliana produced recombinant proteins without -L-fucosidase activity. Pea plants had endogenous -L-fucosidase activity, but the enzyme was not recognised by an antibody produced against recombinant FUC1 protein expressed in E. coli. In contrast, the antibody immunoprecipitated a 20 kDa protein which was inactive. By chromatographic analysis of pea protein extracts, we separated -L-fucosidase-active fractions from the 20 kDa protein fractions. We conclude that the -L-fucosidase activity is not attributable to the 20 kDa FUC1 protein. A new function for fuc1 gene product, now named PIP20 (for protease inhibitor from pea) is proposed.