Demonstration of a COOH-terminal extension on equine lutropin by means of a common acid-labile bond in equine lutropin and equine chorionic gonadotropin

The beta subunits of equine lutropin and equine chorionic gonadotropin were incubated in 0.013 N HCl for 30 min at 110 degrees C and separated into two fragments by reverse-phase high performance liquid chromatography. The amino acid and carbohydrate compositions of both fragments from each subunit were analyzed. The results demonstrated that equine lutropin-beta has a glycosylated COOH-terminal extension that differs only in carbohydrate composition from the COOH-terminal portion of equine chorionic gonadotropin-beta. This is the first demonstration of a glycosylated COOH-terminal extension in a pituitary glycoprotein hormone.     

Surgical correction of Stahl's ear using the cartilage turnover and rotation method

Various operative methods for the correction of Stahl's ear exist. We, however, have devised an alternative "turnover and rotation" procedure. In this procedure, the deformed cartilage itself is used in its original contour to form a new scaphoid fossa, thus simplifying the corrective procedure and establishing a reliable operative method in which reversion to the original state is thought to be highly unlikely.     

Reevaluation of the amino acid sequence of porcine follitropin

We have reevaluated the sequence of porcine follicle-stimulating hormone (pFSH) with more recent protein-sequencing methodology. This has led to revision of the earlier proposed sequence. As with almost all reported gonadotropin -subunits, NH₂-terminal heterogeneity was found in the porcine FSH -subunit (FSH), starting with residue Phe (1), Asp (3), Gly (4), or Thr (7). In the -subunit, there were found to be at least two molecular species, starting with residue Asn (1) (minor 20%) or Cys (3) (major 80%) as NH₂-terminal and ending at residue Glu (108) as COOH-terminal. The net effect of the present revisions is to increase the homology of pFSH with other reported follitropin sequences. Apparent differences in the half-cystine placements in a previous proposal for pFSH compared with other species of FSH are no longer tenable. The half-cystine placements thus remain a constant structural feature throughout the gonadotropin hormones (choriogonadotropin, follitropin, and lutropin).     

Our method of correcting cryptotia

Our technique for the correction of cryptotia using both Z-plasty and the advancement flap is described. The main advantages are the simple design of the skin incision and the possibility of its application to cryptotia other than severe cartilage deformity and extreme lack of skin.     

Isolation, DNA sequence, and regulation of a Saccharomyces cerevisiae gene that encodes DNA strand transfer protein alpha.

DNA strand transfer protein alpha (STP alpha) from meiotic Saccharomyces cerevisiae cells promotes homologous pairing of DNA without any nucleotide cofactor in the presence of yeast single-stranded DNA binding protein. This gene (DNA strand transferase 1, DST1) encodes a 309-amino-acid protein with a predicted molecular mass of 34,800 Da. The STP alpha protein level is constant in both mitotic and meiotic cells, but during meiosis the polypeptide is activated by an unknown mechanism, resulting in a large increase in its specific activity. A dst1::URA3/dst1::URA3 mutant grows normally in mitotic media; however, meiotic cells exhibit a greatly reduced induction of both DNA strand transfer activity and intragenic recombination between his1 heteroalleles. Spore viability is normal. These results suggest that DST1 is required for much of the observed induction of homologous recombination in S. cerevisiae during meiosis but not for normal sporulation.     

Effect of single amino acid substitutions on the protease susceptibility of tryptophan synthase alpha subunit

In order to explore the correlation between protease susceptibility and conformational stability of a protein, the proteolytic degradation by trypsin, subtilisin and pronase P of the wild-type alpha subunit of tryptophan synthase from Escherichia coli and of its two mutant proteins was studied by measuring circular dichroism at 222 nm at various pH values at 37 degrees C. The mutant proteins are substituted by Gln or Met in place of Glu at position 49. The single amino acid substitutions at position 49 significantly affected susceptibility of this protein to the three proteases. Dependence of protease susceptibility of the wild-type and the two mutant proteins on pH was characteristic of each protein and similar for the three proteases. Comparison of the present results with the conformational stabilities of the three proteins previously measured shows that the order of resistance to the proteases among the three proteins coincides with the order of the values of unfolding Gibbs energy change, suggesting that protein degradation depends upon the conformational stability of a protein.     

Localization of axonally transported 125I-wheat germ agglutinin beneath the plasma membrane of chick retinal ganglion cells

The distribution of 125I-wheat germ agglutinin (WGA) transported by axons of chick retinal ganglion cells to layer d of the optic tectum was studied by electron microscopic autoradiography. We found that 52% of the radioactivity was located in axons and axon terminals in the contralateral optic tectum 22 h after intravitreal injection of affinity-purified 125I-WGA. Axons comprised 43% of the volume of layer d. Dendrites, glial cells, and neuron cell bodies contained 20%, 17%, and 3% of the label, whereas these structures comprised 24%, 21%, and 2% of the tissue volume, respectively. We also measured the distances between the autoradiographic silver grains and the plasma membranes of these profiles, and compared observed distributions of grains to theoretical distributions computed for band-shaped sources at various distances from the plasma membranes. This analysis revealed that the radioactive source within axons was distributed in a band of cytoplasm extending in from the plasma membrane a distance of 63 nm. Because WGA is known to bind to specific membrane glycoconjugates, we infer that at least some glycoconjugates may be concentrated within an annular region of cytoplasm just beneath the axonal plasma membrane after axoplasmic transport from the neuron cell body.     

Expression of human immunoglobulin E epsilon chain cDNA in E. coli.

Using the cDNA of human epsilon chain, three expression plasmids that code directly the constant portion of the epsilon chain (C epsilon 1-C epsilon 4, C epsilon 2-C epsilon 4 and C epsilon 3-C epsilon 4 domains) were constructed. These epsilon chain peptides were synthesized in E. coli under the control of the trp promoter-operator. The bacterially produced peptides have the antigenicity of human epsilon chain and gave the molecular weights equal to the values calculated from the amino acid sequence of the constructed plasmids.     

Molecular cloning and nucleotide sequencing of human immunoglobulin epsilon chain cDNA.

DNA complementary to mRNA of human immunoglobulin E heavy chain (epsilon chain) isolated and purified from U266 cells has been synthesized and inserted into the PstI site of pBR322 by G-C tailing. This recombinant plasmid was used to transform E. coli chi 1776 to screen 1445 tetracycline resistant colonies. Nine clones (pGETI - 9) containing cDNA coding for the human epsilon chain were recognized by colony hybridization and Southern blotting analysis with a nick-translated human IgE genome fragment. The nucleotide sequence of the longest cDNA contained in pGET2 was determined. The results indicate that the sequence of 1657 nucleotides codes for 494 amino acids covering a part of the variable region and all of the constant region of the human epsilon chain. Most of the amino acid sequence deduced from the nucleotide sequence is in substantial agreement with that reported. Furthermore a termination codon after the -COOH terminal amino acid marks the beginning of a 3' untranslated region of 125 nucleotides with a poly A tail. Taking this into account, the structure of the human epsilon chain mRNA, except a part of the 5' end, is conserved fairly well in the cDNA insert in pGET2.