Isolation, macromolecular properties, and combining site of a chito-oligosaccharide-specific lectin from the exudate of ridge gourd (Luffa acutangula)

A lectin specific for chito-oligosaccharides from the exudate of ridge gourd (Luffa acutangula) fruits has been purified to homogeneity by affinity chromatography. The lectin has a molecular weight of 48,000, an S(0)20,w of 4.06 S and a Stokes radius of 2.9 nm. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single band corresponding to Mr of 24,000 was observed both in the presence and absence of beta-mercaptoethanol. The subunits in this dimeric lectin are, therefore, held together solely by noncovalent interactions. The lectin is not a glycoprotein, and secondary structure analysis by CD measurements showed 31% alpha-helix. The hemagglutinating activity of L. acutangula agglutinin was not inhibited by any of the monosaccharides tested. Among the disaccharides only di-N-acetylchitobiose was inhibitory. The inhibitory potency of chito-oligosaccharides increased dramatically with their size up to penta-N-acetylchitopentaose. The lectin has two binding sites for saccharides. The affinity of chito-oligosaccharides for L. acutangula lectin, as monitored by titrating the changes in the near UV-CD spectra and intrinsic fluorescence, increased strikingly with the number of GlcNAc units in them. The values of delta G, delta H, and delta S for the binding process showed a pronounced dependence on the size of the chito-oligosaccharides, indicating that the binding of higher oligomers is progressively more favored thermodynamically than di-N-acetylchitobiose. The thermodynamic data are consistent with an extended binding site in this lectin, which accommodates a tetrasaccharide.     

Neuron differentiation and axon growth in the developing wing of Drosophila melanogaster

Sensory neurons in the wing of Drosophila originate locally from epithelial cells and send their axons toward the base of the wing in two major bundles, the L1 and L3 nerves. We have estimated the birth times of a number of identified wing sensory neurons using an X-irradiation technique and have followed the appearance of their somata and axons by means of an immunohistochemical stain. These cells become immunoreactive and begin axon growth in a sequence which mirrors the sequence of their birth times. The earliest ones are born before pupariation and begin axonogenesis within 1 to 2 hr after the onset of metamorphosis; the last are born and differentiate some 12 to 14 hr later. The L1 and L3 nerves are formed in sections, with specific neurons pioneering defined stretches of the pathways during the period between 0 and 4 hr after pupariation (AP), and finally joining together around 12 hr AP. By 16 hr AP the adult complement of neurons is present and the adult peripheral nerve pattern has been established. Pathway establishment appears to be specified by multiple cues. In places where neurons differentiate in close proximity to one another, random filopodial exploration followed by axon growth to a neighboring neuron soma might be the major factor leading to pathway construction. In other locations, filopodial contact between neighboring somata does not appear to occur, and axon pathways joining neural neighbors by the most direct route are not established. We propose that in these cases additional factors, including veins which are already present at the time of axonogenesis, influence the growth of axons through non-neural tissues.     

Cell culture and in vitro studies of fresh and cryopreserved human insulinoma

Summary Dispersed cells from both fresh and cryopreserved human insulinoma have been maintained in cell culture. Initial yield of viable cells was 50% for fresh and 25% for cryopreserved tissue. Viability of cells in culture was documented by increasing numbers of cells (doubling time approximately 5 d initially and 2 d at the sixth subculture for both fresh and cryopreserved tissue) and continued release of insulin over time (approximately 100 ng/ml per 10⁵ cells at 10 d and 175 ng/ml per 10⁵ cells at 30 d of culture for both fresh and cryopreserved tissue). Evidence that cells growing in culture were beta cells was provided by: (a) recovery of intracellular and extracellular immunoreactive insulin (IRI), (b) electron microscopic morphology, and (c) immunohistochemical staining. Cells from fresh insulinoma incubated with increasing concentrations of extracellular glucose released increasing amounts of IRI up to approximately 15 mM glucose, which paralleled changes in plasma insulin obtained during a preoperative glucose tolerance test. Under an Intergovernmental Personnel Act Exchange from the Department of Surgery, University of California, Davis, Sacramento Medical Center.     

Genetic engineering applied to the development of vaccines

The simplest application of the modern genetic manipulation methods to vaccine development is the expression in microbial cells of genes from pathogens that encode surface antigens capable of inducing neutralizing antibodies in the host of the pathogen involved. This procedure has been exploited successfully for development of a vaccine against hepatitis B virus (HBV) that is now widely used. Similar approaches have been directed towards formulations for immunization against several other animal and human diseases and some of these preparations are now presently in trials. Of no less importance is the impact of biotechnology in providing reagents for fundamental studies of topics such as the determination of virulence, antigenic variation, virus receptors and the immunological response to viral antigens. The core antigen of HBV is a good example of a product of genetic engineering that is a valuable diagnostic reagent, and that is finding important use in immunological studies of particular pertinence to vaccine development.