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We isolated a cDNA encoding a novel glucuronyltransferase, designated GlcAT-D, involved in the biosynthesis of the HNK-1 carbohydrate epitope from rat embryo cDNA by the degenerate polymerase chain reaction method. The new cDNA sequence revealed an open reading frame coding for a protein of 324 amino acids with type II transmembrane protein topology. The amino acid sequence of GlcAT-D displayed 50.0% identity to rat GlcAT-P, which is involved in the biosynthesis of the HNK-1 epitope on glycoproteins. Expression of GlcAT-D in COS-7 cells resulted in the formation of the HNK-1 epitope on the cell surface. The enzyme expressed in COS-7 cells transferred a glucuronic acid (GlcA) not only to asialo-orosomucoid, a glycoprotein bearing terminal N-acetyllactosamine structure, but also to paragloboside (lacto-N-neotetraosylceramide), a precursor of the HNK-1 epitope on glycolipids. Furthermore, substrate specificity analysis using a soluble chimeric form of GlcAT-D revealed that GlcAT-D transfers a GlcA not only to Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine++ + but also to Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-pyridylamine++ +. Enzymatic hydrolysis and Smith degradation of the reaction product indicated that GlcAT-D transfers a GlcA through a beta1,3-linkage to a terminal galactose. The GlcAT-D transcripts were detected in embryonic, postnatal, and adult rat brain. In situ hybridization analysis revealed that the expression pattern of GlcAT-D transcript in embryo is similar to that of GlcAT-P, but distinct expression of GlcAT-D was observed in the embryonic pallidum and retina. Regions that expressed GlcAT-D and/or GlcAT-P were always HNK-1-positive, indicating that both GlcATs are involved in the synthesis of the HNK-1 epitope in vivo.

1. Amacrine and ganglion cells in the carp retina were identified from such criteria as photoresponses, intracellular dye staining, responses to optic nerve stimulation and behaviour to a synapse blocking agent. 2. Responses of ganglion cells were accompanied by spike discharges., either facilitated or suppressed by photic stimulation. The cells were also invaded by antidromic impulses, which survived after chemical synapses had been blocked by application of atomized CoCl2 solution. In subsequent histology of the Procion‐stained neurones, the cell bodies were found in the ganglion cell layer and the axons were often traced. 3. Amacrine cells were subdivided into two types. The first type gave rise to transient depolarizations at both on‐ and offsets of spot and annulus illuminations, usually being associated with spike discharges of which the amplitudes varied in different cells. In histology, the cell bodies of this type were situated in the inner nuclear layer and dendrites ramified in two or more discrete sublayers of the inner plexiform layer (the stratified amacrine cell of Cajal). 4. The second type of amacrine cells produced sustained responses during illumination, being associated with no spike but with small oscillatory wavelets. The cell bodies were situated in the inner nuclear layer and the dendrites ramified in a single sublayer of the inner plexiform layer (the monolayered amacrine cell). 5. An attempt was made to see the effect of activation of centrifugal fibres on amacrine cells, but almost all of about 200 cells examined did not respond to optic nerve stimulation. Only two cells produced, with long latency, a small post‐synaptic depolarization which disappeared after chemical synapses in the retina had been blocked. It is considered that the physiological role of the centrifugal system is insignificant in the carp retina.

A wide range of bacterial species, e.g., Enterobacteriaceae and Neisseria, Streptococcus, Branhamella, Haemophilus, Gemella, Pseudomonas, Flavobacterium, and Bacteroides species, were successfully preserved for 1 to 5 years by our gelatin disk drying method. The beta-lactamase activity of penicillinase-producing Neisseria gonorrhoeae was retained for more than 3 years with this method. Good results were also obtained upon airmailing many strains of N. gonorrhoeae embedded in gelatin disks from Japan to the United States. Neisseria, Branhamella, Gemella, and Haemophilus organisms suspended in the reagent used in the preparation of the gelatin disks could be preserved for 6 to 12 months after freezing the cell suspensions at -20 degrees C. Furthermore, modification of our gelatin disk preservation method made possible the safe long- and short-distance transportation of clinical isolates. Our methods can be used by any small laboratory, since they require only conventional instruments and reagents.

Studies of Neisseria gonorrhoeae are difficult to perform because of the organism's poor survival in vitro. To solve this problem we tried to preserve the organism by a gelatin-disc method. The rate of survival and changes of variations in some biochemical properties of eight strains of N. gonorrhoeae were followed for three years. These studies proved that preservation was satisfactory with only a 1/10 reduction of the living cells. Another trial showed that the organism survived for over six months after being frozen at -20 degrees C. The colonial types, agglutination against red cells from rabbit and guinea pig, and antibiotic susceptibility to penicillin, chloramphenicol, tetracycline, kanamycin, and streptomycin did not change after three years' preservation. [ABSTRACT FROM PUBLISHER]