Monoclonal antibody directed against RNA polymerase II of Drosophila melanogaster

Summary Monoclonal antibodies were raised against purified RNA polymerase II (or B) from Drosophila melanogaster. The antibody produced by one hybridoma cell clone was found to be directed against the two large subunits of the enzyme. The absence of antibodies directed against proteins possibly contaminating the antigens used for immunization allowed us to identify RNA polymerase unequivocally in interbands and puffs of polytene chromosomes. Within a single heat shock puff (87C1) RNA polymerase was found to be clustered in two separate areas suggesting two distint regions of RNA polymerase activity in this puff.Abbreviations FITC fluorescein isothiocyanate - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - SDS sodium dodecyl sulfate - Enzyme DNA-dependent RNA polymerase or nucleotide-triphosphate - RNA nucleotidyltransferase (EC 2.7.7.6)     

Trypanosoma cruzi: defined medium for continuous cultivation of virulent parasites.

A liquid medium was developed for the continuous cultivation of Trypanosoma cruzi. Among the several highly purified macromolecules tested only bovine liver catalase, horseradish peroxidase, lactoperoxidase, and bovine hemoglobin supported the continuous growth, at high yield, of mice-virulent Trypanosoma cruzi; other hemoproteins were inactive. Bovine liver catalase showed optimal Trypanosoma cruzi growth-promoting activity, parasites reaching 20 × 10⁶ parasites/ml (95% epimastigotes) at about 10 days in most of the 45 subpassages to date. Furthermore, this protein in the incubation medium provided all the amino acid requirements of actively growing parasites, thus eliminating the need for exogeneous free amino acids. Additional experiments revealed that the hemoprotein's growth-promoting activity was independent of any enzymatic activity and that reconstituting the exact protein composition by means of exogeneous amino acids did not support parasite multiplication, suggesting the importance of the primary structure of the active proteins for growth-promoting activity. These active macromolecules supported the multiplication of five different strains of Trypanosoma cruzi, but did not support Leishmania brasiliensis or Leishmania mexicana proliferation, suggesting species specificity.     

Structural investigation of the capsular polysaccharide of Klebsiella serotype k23

Klebsiella K23 capsular polysaccharide has been investigated by the techniques of hydrolysis, methylation, Smith degradation-periodate oxidation, and base-catalysed degradation, either on the original or the carboxyl-reduced polysaccharide. The structure was found to consist of a tetrasaccharide repeating-unit, as shown below. The anomeric configurations of the sugar residues were determined by ₁H-and ¹³C-n.m.r. spectroscopy on the original and degraded polysaccharides.

     

The synthesis of 1,6-anhydro-β-d-glucopyranose and d-glucosyl oligosaccharides from maltose by a fungal glucosyltransferase

The formation of 1,6-anhydro-β-d-glucopyranose and several d-glucosyl oligosaccharides has been observed during the action of a purified, fungal glucosyltransferase (EC 2.4.1.24) on maltose. Such products are synthesized by a transglucosylation mechanism involving the formation of a d-glucosyl-enzyme complex and the displacement of the d-glucosyl group by appropriate acceptor-substrates. The formation of the 1,6-anhydro bond is a novel type of transfer reaction and occurs by displacement of the enzyme from the d-glucosyl-enzyme complex by the proton of the primary hydroxyl group of the same glucosyl group. This reaction is characterized by inversion of configuration at the position of glucosidic bond-cleavage of the substrate. Synthesis of the d-glucosyl oligosaccharides occurs by displacement of the d-glucosyl groups from the enzyme by suitable acceptor-substrates. In these cases, the reactions are characterized by retention of configuration of the d-glucosidic bonds of the substrate. The list of oligosaccharides produced from maltose includes nigerose, kojibiose, isomaltose, maltotriose, panose, isomaltotriose, and 6-O-d-glucosyl-panose. The identity of these compounds has been established by methylation analysis and enzymic hydrolysis. d-Glucose is also a product of the reaction and arises from both the reducing and the non-reducing groups of maltose.     

Synthesis of nerve growth factor in rat glioma cells

We have analyzed the incorporation of radioactive amino acids by rat C6 glioma cells into material precipitable with anti-β-nerve growth factor (NGF). We show that amino acids are incorporated into a protein the size of β-NGF which is immunologically related to NGF and which has peptides similar to those of mouse β-NGF. Several lines of evidence obtained in this study support the hypothesis that NGF is produced by the proteolytic cleavage of a higher molecular weight precursor. This evidence includes kinetic studies, demonstration of higher molecular weight material (24,000) immunologically related to NGF, and in vitro processing of the 24,000 MW protein to material of the approximate size of β-NGF.     

Endocytosis,digestive vacuolar movement and exocytosis on refeeding starvedTetrahymena pyriformis GL-9

Summary Evidence is presented in support of the hypothesis that the contents of digestive vacuoles in refed starvedTetrahymena pyriformis GL-9 are egested from the cell in approximately the sequence of their order of formation. The investigations involved measurements of the rates of disappearance of digestive vacuoles from the cells and the subsequent appearance of egested globules in the surrounding medium using both cultures and individual cells. The cells were first fed peptone and latex particles for a period and then this type of vacuole formation was suppressed by the addition of excess carmine particles (or the process was repeated with the particles in reverse order). Thus two types of morphologically distinct digestive vacuoles could be produced and observed microscopically. These observations suggest that the temporal nature of the movement of the digestive vacuoles through the cell result in the temporal nature of egestion and that no selective mechanism occurs at egestion. Thus digestive vacuoles are thought to pass through the cell from cytopharynx to cytoproct in approximately the order formed and at approximately constant rate. Under conditions of excess nutrients, where the cells become filled with digestive vacuoles, they seem to be able to maintain an approximately uniform number of digestive vacuoles within themselves by maintaining approximately constant and equal rates of vacuole formation and egestion. The maximum rates of latex or carmine vacuole formation or egestion found in single cells were approximately 0.3–0.4 vacuoles per cell per minute. The results are discussed.     

Citrate lyase from Streptococcus diacetilactis

Citrate lyase (EC 4.1.3.6) was purified 38-fold from cell-free extracts of Streptococcus diacetilactis. The enzyme was homogeneous in analytical ultracentrifugation and polyacrylamide gel electrophoresis The final enzyme preparation contained acetate: HS-citrate lyase ligase—an acetylating enzyme which converts inactive HS-citrate lyase into enzymatically active acetyl-S-citrate lyase. This enzyme activity was purified 25-fold over the crude extract and seemed to be associated with citrate lyase. Partially purified citrate lyase from Leuconostoc citrovorum contained also its acetylating enzyme. Purified citrate lyases from Klebsiella aerogenes and Rhodopseudomonas gelatinosa were devoid of acetylating enzyme activity. The HS-form of citrate lyase from S. diacetilactis was completely acetylated and hence activated by incubation with ATP and acetate for 25 min at 25° C. The enzyme did not acetylate the HS-lyases from R. gelatinosa and K. aerogenes. In contrast to the citrate lyases from R. gelatinosa and K. aerogenes the enzymes from S. diacetilactis and L. citrovorum showed onlya very weak reaction inactivation. It is assumed that this is due to the association of the acetylating enzymes with these lyases.     

Development and diversity of lignin patterns

Different patterns of lignified cell walls are associated with diverse functions in a variety of plant tissues. These functions rely on the stiffness and hydrophobicity that lignin polymers impart to the cell wall. The precise pattern of subcellular lignin deposition is critical for the structure–function relationship in each lignified cell type. Here, we describe the role of xylem vessels as water pipes, Casparian strips as apoplastic barriers, and the role of asymmetrically lignified endocarp b cells in exploding seed pods. We highlight similarities and differences in the genetic mechanisms underpinning local lignin deposition in these diverse cell types. By bringing together examples from different developmental contexts and different plant species, we propose that comparative approaches can benefit our understanding of lignin patterning mechanisms.

Diverse lignin patterns underpin distinct functions in different plant tissues.