Developmental regulation of presence of binding sites for neoglycoproteins and endogenous lectins in various embryonic stages of human lung,liver and heart

Protein-carbohydrate interactions are supposed to play key roles in the mechanisms of cell adhesion, biosignalling and intracellular routing, warranting the analysis of the developmental course of expression of epitopes of this system. Thus, a panel of carrier-immobilized carbohydrate ligands was used as probes, namely lactose,N-acetylgalactosamine,N-acetylglucosamine, mannose, fucose and maltose. Additionally, an antibody to an endogenous -galactoside-binding lectin (anti-galectin-1), the biotinylated lectin and two further human lectins, namely the macrophage migration inhibitory factor-binding sarcolectin and serum amyloid P component (SAP) that displays selectivity for sulphated sugars and mannose-6-phosphate, were included. They enabled us to assess the extent of the presence of respective binding sites in fixed sections from human lungs (pulmonary epithelial cells), livers (hepatocytes) and hearts (myocard cells) of 10–50 weeks gestation. Invariably, specific binding was detected in the three organ types, at least in certain stages. In most of the cases, the intensity of staining exhibited developmental regulation. The apparent patterns reveal similarities between the different cell types, as seen with immobilizedN-acetylglucosamine as well as with labelled galectin-1 and sarcolectin. However, drastic differences among such patterns with nearly opposite developmental courses do also occur, as detected for carrier-attached mannose and maltose residues. These results point to a potential importance for the detected glycohistochemical features in human development and substantiate the possibility of differential regulation of the presence of binding sites for distinct sugars within a certain organ and between the individual cell types of the monitored organs.     

Comparison between maize root cells and their respective regenerating protoplasts: wall polysaccharides

The cell-wall polysaccharides from different parts of maize roots have been analysed. The arabinose, galactose and mannose contents are influenced by cell differentiation, whereas xylose, rhamnose and uronic-acid contents are not. In cap cells, the pectin content is low but rhamnose and fucose are present in larger quantities. The cell-wall polysaccharides from cells of the elongation zone and their respective regenerating protoplasts were also analysed. The walls of the protoplasts contained higher xylose and mannose levels and a much lower level of cellulose than the cells from which they were derived.     

The paramedial neurosecretory cells of the suboesophageal ganglion of the cricket,Teleogryllus commodus (Walk.)

Summary Ovariectomy, performed immediately after the final hatch, caused a reduction of stainable (neurosecretory?) material in the paramedial neurosecretory cells (PNC) (A-type) of the suboesophageal ganglion in 10 day-old females of Teleogryllus commodus (Walk.). A concomitant increase in nuclear volume and in the incorporation of ³⁵S-cysteine indicates increased synthesis of neurosecretory material. From these findings it is concluded that more stainable material is secreted in the cerebral neurohaemal organ after Ovariectomy. A functional relationship between the PNC and the ovaries is suggested.     

Subcellular distribution of enzymes involved in α-glucan utilization in Klebsiella pneumoniae

The double-isotopic labelling technique was used to identify comprehensively proteins involved in α-glucan catabolism in Klebsiella pneumoniae NCTC 9633. Cells were grown with either glycerol in the presence of ³H-leucine or with glycerol plus maltose in the presence of ¹⁴C-leucine. Each labelled culture was then fractionated into the main subcellular components, i.e. the cytoplasm, periplasm, cytoplasmic and outer membrane. Corresponding fractions derived from ³H-labelled and ¹⁴C-labelled cells were combined, and the proteins were analyzed by polyacrylamide gel electrophoresis under denaturing conditions. Gel slices were then counted for ³H- and ¹⁴C-radioactivity, a positive deviation from the standard ¹⁴C/³H ratio being evidence for the presence of a protein specifically induced by maltose in the culture medium. The protein pattern thus obtained was compared with the properties of proteins comprising a similar pathway for maltodextrin utilization in Escherichia coli K-12. Ample information which has been obtained mainly by genetic analysis is available about maltodextrin-utilizing enzymes in E. coli K-12.

1. Cytoplasm. Neither amylomaltase nor maltodextrin phosphorylase, well-known soluble enzymes, were identifiable by the double-labelling technique, presumably because these enzymes constitute only a very minor portion of all soluble proteins in the cytoplasm.
2. Periplasm. A prominent protein with a mass of 43000 daltons (43 kD) was found similar to the maltose-binding protein of E. coli K-12 (44 kD).
3. Cytoplasmic membrane. At least 2 proteins with a mass between 40 and 50 kD were detected, minor proteins were seen at ≈ 15 and ≈ 20 kD. One or 2 of the proteins may function as a permease catalyzing the active transport of maltodextrins.
4. Outer membrane. The major protein had a mass of 55 kD, other proteins were found with ≈ 18, ≈48, and ≈140 kD. The major protein may have the same function as the maltodextrin pore protein in E. coli K-12 (55 kD), because K. pneumoniae could grow on 10 μM maltose at practically the same rate as on 10 mM maltose. The 140 kD protein is pullulanase.

     

Electrical properties of chloride transport across theNecturus proximal tubule

Summary The chloride conductance of the basolateral cell membrane of theNecturus proximal tubule was studied using conventional and chloride-sensitive liquid ion exchange microelectrodes. Individual apical and basolateral cell membrane and shunt resistances, transepithelial and basolateral, cell membrane potential differences, and electromotive forces were determined in control and after reductions in extracellular Cl^–. When extracellular Cl^– activity is reduced in both apical and basolateral solutions the resistance of the shunt increases about 2.8 times over control without any significant change in cell membrane resistances. This suggests a high Cl^– conductance of the paracellular shunt but a low Cl^– conductance of the cell membranes. Reduction of Cl^– in both bathing solutions or only on the basolateral side hyperpolarizes both the basolateral cell membrane potential difference and electromotive force. Hyperpolarization of the basolateral cell membrane potential difference after low Cl^– perfusion was abolished by exposure to HCO ₃ ^– -free solutions and SITS treatment. In control conditions, intracellular Cl^– activity was significantly higher than predicted from the equilibrium distribution across both the apical and basolateral cell membranes. Reducing Cl^– in only the basolateral solution caused a decrease in intracellular Cl^–. From an estimate of the net Cl^– flux across the basolateral cell membrane and the electrochemical driving force, a Cl^– conductance of the basolateral cell membrane was predicted and compared to measured values. It was concluded that the Cl^– conductance of the basolateral cell membrane was not large enough to account for the measured flux of Cl^– by electrodiffusion alone. Therefore these results suggest the presence of an electroneutral mechanism for Cl^– transport across the basolateral cell membrane of theNecturus proximal tubule cell.     

Physiological factors determining formation of plastocyanin and plastidic cytochrome c-553 in Scenedesmus

Physiological conditions necessary for the formation of plastocyanin and the concurrent cessation of cytochrome c-553 formation were studied in cells of copper-deficient Scenedesmus acutus after the addition of copper. Plastocyanin is formed after a lag-phase, leaving constant the content of plastidic cytochrome c-553. Therefore, the concentration of plastocyanin per cell increases and the concentration of cytochrome c-553 decreases during growth. Formation of plastocyanin during the induction period studied is dependent on light intensity. In the dark, there is a 90% inhibition, whereas under light intensities above 50 Wm^-2, a ratio of 1.3 molecules plastocyanin per 1,000 molecules chlorophyll is attained.Plastocyanin formations is inhibited by the uncoupler carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), but not by moderate concentrations of 3-[3,4-dichlorophenyl]-1,1-dimethylurea (DCMU), and by keeping the algae under a nitrogen atmosphere without CO₂. Concurrently, the cultures treated with FCCP show a decreased endogenous ATP level. The ATP is necessary for plastocyanin formation.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - DCMU 3-[3,4-dichlorophenyl]-1,1-dimethylurea - pcv packed cell volume     

Comparison of isolated peanut agglutinin receptor glycoproteins from human,bovine and porcine erythrocyte membranes

Affinity chromatography has been used to isolate and compare the peanut agglutinin receptors from neuraminidase-treated human, bovine and porcine erythrocyte membranes. Passage of Triton X-100-solubilised membrane material through either Sepharose- or acrylamide-based affinity columns resulted in the reversible binding of receptor molecules to the immobilised lectin. Elution with 0.2M galactose released specifically bound glycoprotein fractions, the composition and molecular weights of which were determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.Carbohydrate analysis by gas chromatography identified these bound glycoprotein fractions as the major sources of the $\text{O-}\text{glycosidic-linked}$ disaccharide $\text{galactosyl}\text{-β-(1 → 3)-N-}\text{acetylgalactosamine}$ in these membranes. It is suggested that these isolated fractions represent a discrete population of glycoproteins within the membranes studied, which possess both $\text{O-}\text{glycosidic}$- and $\text{N-}\text{glycosidic-linked}$ carbohydrates.     

Conformation of sequential and random lysine–phenylalanine copolypeptides

The sequential copolypeptides (Lys-Phe-Lys)_n and (Lys₂-Phe-Lys)_n and a series of related random copolypeptides were investigated with respect of their ability to adopt the α-helix or β-conformation. Conformational transitions were induced by increasing the pH or by addition of NaClO₄ or methanol and were observed by recording the CD spectra. In contrast to the respective alternating copolypeptide (Phe-Lys)_n with its strong tendency for the β-structure reported previously, (Lys-Phe-Lys)_n can adopt either secondary structure, whereas (Lys₂-Phe-Lys)_n strongly favors the α-helix. Together with the random copolypeptides, whose composition varied from 20 to 50 mol % phenylalanine and whose average molecular weights ranged from 10,000 to 90,000, the influence of the phenylalanine content and of the chain length on conformational stability and the rotatory strength of the respective secondary structures were elaborated.     

A monoclonal antibody to viral glycoprotein blocks virus-immune effector T cells operating at H-2Dd but not at H-2Kd1.

A particular monoclonal antibody that binds to the influenza virus HA molecule inhibits HA-specific thymus-derived lymphocytes mediating cytotoxicity in the context of H-2Dd but not of H-2Kd. Another monoclonal antibody blocks both sets of HA-specific effector T cells. This observation, together with related findings from other laboratories, is considered to support the idea that T cell recognition is directed against some association of viral and H-2 glycoproteins, as proposed in the original formulation of the "altered self" concept.