Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE

Homopolymeric α-2,8-linked sialic acid (PSA) has been found as a capsular component of sepsis- and meningitis-causing bacterial pathogens, and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5′ end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS-PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C-terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.     

Rapid analysis of amino acids using pre-column derivatization

A new approach to the pre-column derivatization and analysis of amino acids is described. The method is based upon formation of a phenylthiocarbamyl derivative of the amino acids. The derivatization method is rapid, efficient, sensitive, and specific for the analysis of primary and secondary amino acids in protein hydorlyzates. The liquid chromatographic system allows for the rapid, bonded-phase separation with ultraviolet detection of the common amino acids with 12-min analysis time and a 1-pmol sensitivity.     

Resonance Raman resolution of a-, b- and c-type cytochromes in membrane vesicles of alkalophilic bateria

Resonance Raman spectroscopy has been used to obtain complete spectra of each individual cytochrome type — a, b and c — in the reduced state within membrane vesicle preparations from two species of obligately alkalophilic bacteria: Bacillus alcalophilus and Bacillus firmus RAB. The vibrational spectra, in the range 250–1700 cm^?1, were obtained with tunable dye laser excitation in the wavelength range 550–600 nm tuned to resonance with the appropriate reduced alpha band maximum for the cytochrome type of interest. The spectra reveal details which serve to characterize the specific type of cytochrome as well as to confirm the similarity of the heme prosthetic group to previously well-characterized cytochromes of the the a- b- or c-type. Preliminary evidence in support of heterogeneity of b-type, and possibly a-type cytochromes, or of heme-heme interaction within the membrane is presented.     

Growth regulation in Hydra: Relationship between epithelial cell cycle length and growth rate

The relationship between epithelial cell production and growth rate was investigated in Hydra attenuata under different feeding regimes. The increase of epithelial cell number was compared to the duration of the epithelial cell cycle using standard methods of cell cycle analysis. The results indicate that cell cycle changes accompanying changes in feeding regime are not sufficient to explain the altered growth rate. Under heavy feeding regimes, epithelial cell production equals tissue growth rate. At low feeding level or under starvation conditions the epithelial cell cycle lengthens and growth rate of epithelial cell population is slowed. However, the cell cycle changes are insufficient to account for the reduction in tissue growth and thus there is an effective overproduction of epithelial cells amounting to 10% per day. Evidence suggests that these excess cells are phagocytized by neighboring cells in the tissue. Thus phagocytosis is directly or indirectly involved in regulating the growth of hydra tissue.     

Anucleolate frog embryos contain ribosomal DNA sequences and a nucleolar antigen

Previous studies of Xenopus laevis embryos homozygous for the nucleolar deletion mutation have concluded that these embryos contain few, if any, copies of the genes for the 18 S and 28 S ribosomal RNAs. Using hybridization to restriction endonuclease digests of DNA it is found, in fact, that a small amount of ribosomal DNA is still present in such embryos. The ribosomal DNA in these embryos appears to include a few normal repeats together with a variety of unusual fragments containing either spacer or gene sequences. An antibody found in the serum of a scleroderma patient reacts with an antigen localized in the nucleoli of wild-type embryos. In anucleolate embryos this antigen is found in the so-called pseudonucleoli and in many small bodies in the nuclei.     

Radiochemical synthesis of DL-canaline and the colorimetric assay of canaline

A procedure is available for the production of DL-[carboxy-14C]canaline from [14C]cyanide by reaction of ethyl N-hydroxyacetimidate and acrolein to form ethyl N-[3-oxopropoxy]acetimidate. The reaction product is converted to the nitrile and then to the hydantoin derivative of DL-canaline; alkaline hydrolysis produces the free amino acid (2-amino-4-aminooxypropionic acid). This procedure can be extended to the production of DL-[carboxy-14C]canavanine by guanidination of C-1-labeled DL-canaline with O-methylisourea. A markedly improved colorimetric assay for canaline has been achieved by a procedure involving carbamylation of canaline with cyanate to form O-ureidohomoserine (2-amino-4-ureidooxybutyric acid). Colorimetric analysis of the latter amino acid markedly enhances the sensitivity, reproducibility, and accuracy of the analysis of L-canaline from biological materials.     

Exudate-feeding byCallithrix jacchus penicillata in semideciduous woodland (Cerradão) in central Brazil

We report the exudate feeding behavior of two groups of marmosets (Callithrix jacchus penicillata) living permanently in Cerradão, a common woodland formation of Central Brazil. Cerradão is an open canopy formation and marmosets must occasionally descend to the ground in order to move from tree to tree. Even in atypical habitat, exudate eating is the predominant foraging activity. Marmosets are engaged in exudate collection over 70% of the total time spent feeding. They were observed gnawing on seven species of trees, and consumed exudates from four of these species. We compared the degree of utilization of the exudate sources, and examined a number of different characteristics of the exudates. Morphological adaptations that allow for the exploitation of the “exudate-eater niche” may be an important component of the adaptability ofCallithrix marmosets.