Expression of anchorin CII, a collagen-binding protein of the annexin family, in the developing chick embryo.

Expression of anchorin CII, a collagen-binding protein of the annexin family, was followed in the developing chick embryo using Northern and in situ hybridization and Western blotting. During chick somite development, anchorin CII mRNA was detected by Northern blotting as early as stage 11. At stage 24, anchorin mRNA accumulated in the anterior part of the somite sclerotome near the resegmentation line, as shown by in situ hybridization. The presence of anchorin CII protein during stages 11 to 20 was confirmed by Western blotting. In situ hybridization identified anchorin CII also in the otic vesicle adjacent to the site of contact with the statoacoustic ganglion and in the mandibular mesenchyme. The level of anchorin CII mRNA in differentiated hyaline cartilage, exemplified by sternal cartilage, was lower than that in differentiating somites or cultured chondrocytes. These findings are consistent with our notion that anchorin CII may be involved in cell-matrix interactions preceding chondrogenic differentiation events in the chick embryo. A significant level of anchorin CII mRNA and protein synthesis was also found in cultured myoblasts, but less than that in chondroblasts. This distribution pattern is different from that reported for a related protein, p34, or calpactin, the major protein substrate for tyrosine kinase phosphorylation in chick chondrocytes and fibroblasts. The results confirm suggestions from previous sequencing studies that anchorin CII and p34 are different proteins of the annexin/calpactin family.     

Synthetic peptides anchor T cell-specific TNP epitopes to MHC antigens.

Several TNP-specific, H-2Kb-restricted mouse CTL clones were identified which specifically lysed target cells in the presence of tryptic digests of TNP-modified BSA. Glutaraldehyde fixation of cells revealed that the tryptic fragments did not require further cellular processing. Chromatographic fractionation of digested TNP-BSA identified the peptide TNP-BSA222-231, containing a TNP-modified lysine at BSA position 227, as the antigenic entity. The corresponding synthetic peptide was immunologically cross-reactive with the digest. All clones reactive with TNP-BSA222-231 cross-reacted with a similar peptide from mouse serum albumin (TNP-MSA126-135), favoring the assumption that TNP-BSA222-231 represents an artificial determinant, cross-reacting with some as yet unidentified, TNP-modified, Kb-associated self-peptides. Some of our clones also cross-reacted with tryptic digests of TNP-OVA or TNP-keyhole limpet hemocyanin. We interpret these findings to indicate that 1) a significant proportion of hapten (TNP) determinants for T cells are anchored to MHC via peptides; and 2) the amino acid sequence of these peptides may only partly define the specificity of the T cell-relevant hapten epitope, implying a particularly repetitive nature of these determinants. The production of T cell-antigenic hapten-peptide conjugates will hopefully open new roads to study immune responses to environmental allergens.     

Expression and secretion of pea-seed lipoxygenase isoenzymes in Saccharomyces cerevisiae

Summary Lipoxygenases (EC 1.13.11.12) catalyse the oxygenation of polyunsaturated fatty acids such as linoleic and arachidonic acid into reactive cis/trans hydroperoxidiene intermediates, which then serve as substrates for other enzymes leading to the production of a variety of secondary metabolites. In order to explore the characteristics of the individual lipoxygenase isoenzymes in more detail larger amounts of the pure enzymes are needed and their production in a heterologous host is therefore desirable. Full-length cDNAs encoding pea-seed lipoxygenase isoenzymes 2 and 3 were expressed in Saccharomyces cerevisiae with the aid of yeast-Escherichia coli shuttle vectors. Expression of the cDNA for lipoxygenase 2 under the control of the constitutive phosphoglycerate kinase (PGK) gene promoter yielded significant amounts of active enzyme inside the cell, both with yeast transformants carrying the cDNA gene on high-copy-number plasmids or integrated in chromosome V. Addition of the yeast invertase signal sequence in front of the pea lipoxygenase 3 yielded secreted active pea-seed lipoxygenase in the medium, but large amounts of inactive lipoxygenase 3 remained inside the yeast cell. Expression of the LOX3 cDNA can be achieved either constitutively with the PGK promoter or inducibly with the GAL1 promoter. Correspondence to: B. Knust     

Genetic and biochemical characterization of a new chymotrypsin isozyme of the house mouse,CTRA-1

Genetic variation of a codominantly inherited pancreas protease, designated CTRA-1, was discovered in the house mouse by isoelectric focusing in polyacrylamide gels. Phenotype CTRA-1A was found in MOLH/Fre and in the majority of common laboratory mouse strains. Phenotype CTRA-1B was found in PWD/Ph. It was characterized by the absence of a corresponding protease band. A third phenotype, CTRA-1C, was observed in IS/Cam and a fourth phenotype, CTRA-1D, was detected in SEG/1. CTRA-1 was found only in the pancreas and may represent the A form of chymotrypsin. The enzyme was shown to be controlled by the presumed structural locus Ctra-1 located on chromosome 8. From two backcross series, including a total of 274 animals, the gene order (Es-1, Es-9)-3.9 +/- 1.7%-Got-2-3.9 +/- 1.7%-(Es-2, Es-7, Es-23)-0.7 +/- 0.5%- Ctra-1-6.3 +/- 2.2%-Prt-2 was established.     

Structural characterization of glycoprotein carbohydrate chains by using diagoxigenin-labeled lectins on blots

The carbohydrate structures of blotted glycoproteins can be analyzed by probing them with lectins. Here we describe a method where lectins conjugated with digoxigenin are used in combination with an anti-digoxigenin antibody AP conjugate as a very sensitive detection system for this type of analysis. The specificity of the lectins used, and the sensitivity of the detection system, provide valuable conclusions on the glycan structures. Only small amounts of glycoproteins are required for the analysis. The binding specificity of a set of lectins is demonstrated with various glycoproteins of defined carbohydrate structure. The application of these labeled lectins in combination with specific glycosidases for the characterization of the carbohydrate chains of recombinant tissue plasminogen activator and erythropoietin is presented.     

Postrhinoplasty "red nose": differential diagnosis and treatment by laser

Prior to anticipated nasal surgery, the nasal and facial skin should be examined for any vascular lesions. The skin type should be ascertained. A history of any prior nasal surgery, particularly on the nasal dorsum, should be noted. If rosacea is a clinical possibility, a trial of 1.5 to 2.0 gm q.d. of tetracycline for 6 to 8 weeks is warranted. If, after rhinoplasty, a diffuse "redness" on the nasal dorsum results and one can exclude other diagnoses, then argon laser therapy should be considered. A 3-mm punch biopsy should be obtained to see whether superficial ectatic vessels are present, a finding that would be indicative of a good result from laser therapy.     

Characterization of cloned murine cytolytic T cell lines

Murine cytolytic T lymphocytes can be kept in continuous culture apparently indefinitely by repeated passage in a concanavalin A-induced growth promoting medium. Some of these long-term cell lines maintain their cytolytic activity. Starting from three such populations, several cloned cytolytic T cell lines were derived and subsequently subcloned one or more times. Considerable variation in the levels of cytolytic activity was observed between different subclones; some initially active subclones lost activity with prolonged culture. In addition, one of the clones appeared to progressively lose the relative specificity demonstrated during the earlier passages of the parent cell line.     

Ultrastructure of reticulum cells in the bone marrow

In this study the attempt was made to classify the reticulum cells of the bone marrow on the basis of electron-microscopic findings. The basis of the differentiation was the ability of the cells to phagocytize substances or not. For two cell types the intracytoplasmic filaments were used as distinctive marks. The following classification resulted: (a) phagocytic reticulum cells, (b) undifferentiated reticulum cells, (c) fibrous reticulum cells of type I, which contain filaments of 4-8 nm diameter and are located near the blood sinus of the bone marrow, (d) fibrous reticulum cells of type II, which contain intracytoplasmic filaments of 10 nm diameter; since these cells contain neutral fat bodies, the possibility of a reversible conversion to fat cells has to be assumed and (e) fibroblasts, cells which synthesize the substance of the extracellular space. A connection of reticulum cells to haematopoietic functions or to stem cell functions could be found.