A sensitive method to introduce membrane-bound proteins into recipient cells based on affinity enrichment of lipid vesicles to the recipient cell prior to fusion

A sensitive analytical procedure for studying membrane-bound structures has been developed. Membrane glycoproteins inserted into liposomes were transferred to recipient cells by use of a lectin, concanavalin A, bound to the cells as a bridge to generate proximity between the recipient cell and the glycoprotein-containing liposome, prior to exposure to the fusing agent, poly(ethylene glycol). Partially purified histocompatibility antigen from rats was introduced into the membrane of human lymphocytes. After treating the cells with poly(ethylene glycol) under fusion conditions, some of the antigen present in the preparation could not be eluted with alpha-methyl mannoside and EDTA, indicating that incorporation in the cell membrane had taken place. This antigen remained exposed on the lymphocyte surface for approximately 1 h as demonstrated by sensitivity of the lymphocytes to the lytic effect of an antiserum to the histocompatibility antigen in the presence of complement. Some of the lectin molecules seemed to be internalized in the cells but no induction of cell mitosis was observed. The described method gives an opportunity to work with small amounts of membrane proteins inserted into liposomes, introducing them into recipient cells for analysis of their biological activities.     

Isolation and characterization of a heparin with high anticoagulant activity from Anomalocardia brasiliana

The isolation, some structural features, physicochemical properties and pharmacological activities of a heparin from Anomalocardia brasiliana are reported. It is shown that the mollusc heparin is very similar to those present in mammalian tissues with regard to chemical composition, physicochemical properties, pharmacological activities and susceptibility to heparinase and heparitinase II from Flavobacterium heparinum, as well as to the types of products formed by the action of these enzymes. Three significant quantitative differences were observed for the mollusc heparin when compared with the ones from mammalian origin, namely, a higher degree of binding with antithrombin III (45%), higher molecular weight (27-43 kDa) and higher anticoagulant activity (320 I.U./mg). The possible biological role of heparin is discussed in view of the present findings.     

Effect of the rehydration medium on the recovery of freeze-dried lactic acid bacteria.

Sixteen cultures of lactic acid bacteria were freeze-dried in 10% nonfat skim milk plus 0.75 M adonitol and rehydrated by using different rehydration media. Marked variations in their capacity to repair cellular damage after freeze-drying were observed among the species and strains under consideration.     

Chloride as allosteric effector of yeast aminopeptidase I

Activation of yeast aminopeptidase I by chloride was studied by kinetic methods. Several effects contributed to overall activity enhancement: At low concentrations of Zn2+ (an essential component of aminopeptidase I) chloride increased the amounts of active enzyme by reducing the cooperativity of metal binding. In addition, substrate turnover was enhanced due to increased kcat and a moderate decrease of Km. At high concentrations of Zn2+ substrate saturation curves were sigmoidal. Under these conditions chloride activated by restoring Michaelis-Menten kinetics of substrate turnover. At the same time, reconstitution of active enzyme from apoprotein and Zn2+ was substantially accelerated and its inactivation due to loss of Zn2+ was retarded. Co2+-Substituted aminopeptidase I, although catalytically active, was much less sensitive to chloride activation. Apparent binding constants for chloride, as estimated from its effects on metal binding and catalysis, respectively, were different. This suggests that two independent activation mechanisms may be operative. Both appear to be mediated by conformational changes of the enzyme protein.     

Changes in the mucosa of the duodenum and stomach as a result of experimental duodenal ulcers and vagotomy

By means of the transmissive and scanning electron microscopy methods and radioautography, structure of mucous membrane of the stomach and duodenum has been studied under experimentally induced duodenal ulcers before and after vagotomy during various time. The vagotomy results in accelerated healing of the ulcer defect. This is connected with an increased proliferative activity in the crypta cells, however, this is accompanied with deceleration of their differentiation. Under the duodenal ulcers the amount of chief and parietal cells increases in the gastric mucous membrane, this depends on gastrostasis produced by stenosis of the pylorus. At vagotomy the amount of the chief and parietal cells in the fundal glands of the mucous membrane decreases; this is accompanied with a lowered secretory activity.     

Structure of an antifreeze polypeptide and its precursor from the ocean pout, Macrozoarces americanus

Serum antifreeze polypeptides (AFP) from Newfoundland ocean pout have been resolved by ion exchange chromatography and reverse phase high performance liquid chromatography into at least 12 components. The protein sequences of three of the AFP were determined using a combination of protein Edman degradation and cDNA sequencing. The AFP precursor protein encodes for a preprotein of 87 amino acids with no obvious prosequences. Two of the AFP (SP1-A and SP1-C) were separate gene products with minor amino acid sequence differences. The protein structure of SP1-C precursor is MKSVILTGLLFVLLCVDHMTASQSVVAT QLIPINTALTPAMMEGKVTNPIGIPFAEMSQIVGKQVNTPVAKGQTLMPNMVKTYVAGK. The third AFP (SP1-B) is a post-translation modification product of SP1-C. These experiments indicate that the ocean pout AFP are a multigene family with protein structure different from any other known polypeptide antifreezes.     

The orientation of the distal part of the quadriceps femoris muscle as a function of the knee flexion-extension angle

Lateral view radiographs of ten autopsy knees were used to determine the orientation of the patellar ligament, patella and quadriceps tendon relative to tibia and femur at different flexion-extension angles (0-120 degrees) of the knee. The results show a linear relationship between the angle of flexion and the movement of the patellar ligament relative to the tibia and of the movement of the patella relative to tibia and femur. There is a non-linear relationship between angle of flexion and the movement of the quadriceps tendon relative to the patellar ligament, patella and femur. The angular changes between patella and patellar ligament are negligible. The complicated movements of the distal part of the quadriceps femoris muscle may significantly influence biomechanical parameters such as the forces acting at the patella and tibial tuberosity.     

2H NMR investigation of the organization and dynamics of polyisoprenols in membranes

The polyisoprenols (PIs) dolichol and undecaprenol function as chemical carriers of glycosyl residues in the membrane-directed synthesis of glycoconjugates in prokaryotic and eukaryotic cells. The molecular details of how these lipid cofactors function is unknown. Presented here are results of deuterium NMR investigations of site specifically 2H-labeled PIs incorporated into model membranes. To complement previous omega-terminal PI labeling schemes, a simple synthesis of head group 2H-labeled PIs is presented in which a PI alcohol is esterified with deuterated acetyl chloride. The 2H-labeled PIs, when incorporated into multilamellar membranes composed of phosphatidylcholine, gave rise to 2H NMR powder patterns interpretable in terms of quadrupole splittings (delta vQ) and spin-lattice relaxation times (T1s). Pure isomers of head group 2H-labeled geraniol (C10) and solanesol (C45) gave rise to single splittings while farnesol (C15) gave rise to two sets of splittings due to cis-trans isomerization at the polar terminal double bond. Membranes containing C45 solanesol exhibited a large isotropic component, indicative of limited partitioning of this poly trans PI into the membrane. T1 measurements revealed high rates of motion for PIs relative to cholesterol in similar membrane hosts and revealed correlation times close to the fatty acyl methyl termini in phosphatidylcholine. The smaller PIs showed higher rates of motion but the T1s of head and tail labels were similar. These data indicate that both ends of the esterified PI molecules see similar environments, probably in the bilayer interior, and suggest that the esterified PIs studied here do not appear to adopt a conventional head group-at-interface orientation of lipids within the bilayer.     

Biosynthesis of the neural cell adhesion molecule: characterization of polypeptide C

The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B and a 115,000 Mr polypeptide C, whereas neurons expressed a 200,000 Mr polypeptide A as well as polypeptide B. Skeletal muscle cells produced polypeptide B. The polypeptides synthesized by the three cell types were immunochemically identical. The membrane association of polypeptide C was investigated with methods that distinguish peripheral and integral membrane proteins. Polypeptide C was found to be a peripheral membrane protein, whereas polypeptides A and B were integral membrane proteins with cytoplasmic domains of approximately 50,000 and approximately 25,000 Mr, respectively. The affinity of the membrane binding of polypeptide C increased during postnatal development. The posttranslational modifications of polypeptide C were investigated in glial cell cultures, and it was found to be N-linked glycosylated and sulfated.     

An immunological approach to enrich a mitotic stimulator and to reveal G2-phase-specific proteins in Physarum polycephalum

Purified antibodies from an antiserum against S-phase proteins of the myxomycete Physarum polycephalum were attached to protein-A-Sepharose CL-4B. A late G2-phase extract that contained a mitosis-stimulating protein was applied to this immunoadsorbent, and the mitosis-stimulating protein was enriched by a factor of ten. This protein, which is present in the cell in low amounts, is synthesized in late G2 phase and obviously degraded in a later stage of the cycle. Immunoadsorption of a G2-phase extract with anti-S-antibodies decreased the 700 main proteins to 20 as demonstrated by two-dimensional gel electrophoresis. No difference in protein pattern could be observed on two-dimensional gels between S-phase and G2-phase extracts before and after immunoadsorption with anti-S-antibodies. This indicates that there are no G2-phase-specific proteins among the 700 most abundant proteins of Physarum polycephalum.