Isolation and analysis of monoclonal antibodies against various gangliosides

Female BALB/c mice were immunized with human melanoma (Mewo) cells containing ganglioside GD3 as a surface antigen. Immune splenocytes were fused with syngeneic P3-X63.Ag 8 myeloma cells. Antibodies produced by hybrid clones were analyzed by solid phase immunoassay. B, C, D and Q clones producing antibodies against Raja clavata brain gangliosides were obtained. Monoclonal B and C antibodies bound monosialogangliosides. Monoclonal D antibody bound a number of gangliosides but reacted predominantly with GD1a. Monoclonal Q antibody reacted selectively with GQ1c. It is assumed that ganglioside GQ1c is expressed on the melanoma cell surface and may be found only in the early stage of ontogenesis of high vertebrates.     

Modification of testicular and thyroid function by chronic exposure to short photoperiod: a comparison in four rodent species

Exposure of male Syrian hamsters (Mesocricetus auratus) for 10 weeks to short photoperiod (SP) providing 10 hr light: 14 hr darkness (10:14 LD) produced a significant reduction in the weights of the reproductive organs, plasma thyroxine (T4) levels and free T4 index (FT4I) compared to the values of animals exposed to long photoperiod (LP, 14:10 LD). C57bl male house mice (Mus musculus) kept in SP (10:14 LD) had reproductive organ weights equivalent to those of mice kept in long days (14:10 LD) and lower T3 uptake (T3U) values. Male gerbils (Meriones unguiculatus) exposed to 13 weeks of SP (10:14 LD) had lower body weights, testes and seminal vesicle weights and higher T3U values compared to LP (14:10 LD) controls. However, no effect was seen on plasma T4 and triiodothyronine (T3) values nor the FT4I and free T3 index (FT3I). White-footed male mice (Peromyscus leucopus) exposed to SP (8:16 LD) had significantly lower testes and seminal vesicle weights while plasma T4 and T3 levels were unaffected. Snell strain house mice (Mus musculus) exposed to SP (8:16 LD) had normal reproductive organ weights compared to the values of LP-exposed (16:8 LD) control animals. However, there was a significant depression in T3 and in the FT3I in the SP animals.     

Perturbation of growth and differentiation of Friend murine erythroleukemia cells by 5-bromodeoxyuridine incorporation in early S phase.

Cultured Friend murine erythroleukemia cells (Friend cells) are induced to undergo erythroid differentiation when grown in the presence of dimethylsulfoxide (DMSO) and other compounds. The effects of unifilar substitution of bromouracil (BU) for thymidine in the DNA (BU-DNA) of Friend cells were examined. Cells were grown in the presence of 5-bromodeoxy-uridine (BrdU) for one generation, then centrifuged and resuspended in medium containing DMSO without BrdU. These cells exhibited a delay in the appearance of heme-producing, benzidine-reative (B+) cells and a decreased rate of cell proliferation in comparison to the control not containing BU-DNA. A transient inhibition of entry into S phase was observed when control cells or cells containing BU-DNA were grown in the presence of DMSO) for 10 to 20 hours. This transient inhibition was increased in the BrdU culture. Thus BU-substitution in Friend cells alters other cellular functions in addition to erythroid differentiation. The rate of increase in the percent of cells committed to differentiate (those forming B+ colonies in plasma clots) was similar in the BrdU and control cultures until 40 to 50 hours. After this time, a delay in the appearance of committed cells was observed in the BrdU culture. The effect of BrdU on the appearance of B+ cells was more pronounced and occurred earlier than its effect on the rate of commitment. Therefore, the delay in the appearance of B+ cells in the BrdU culture was due primarily to perturbation of post-commitment events such as the accumulation of hemoglobin. We also examined the effect on growth and differentiation after BrdU was incorporated during different intervals of S phase in cells synchronized by centrifugal elutriation or by double thymidine block and hydroxyurea treatment. The delay in the appearance of B+ cells and inhibition of cell proliferation were only observed when BrdU was incorporated in the first half of S phase. BrdU (10 muM) had no effect on growth or differentiation when present during late S or G1 and G2. These results, using two very different methods to achieve cell synchrony, indicate that the effects of BrdU on growth and differentiation described above are due to its incorporation into DNA sequences replicating during early S.     

Solid-phase synthesis of polymyxin B1 and analogues via a safety-catch approach.

As part of a program towards the development of novel antibiotics, a convenient method for solid-phase synthesis of the cyclic cationic peptide polymyxin B1 and analogues thereof is described. The methodology, based on cleavage-by-cyclization using Kenner's safety-catch linker, yields crude products with purities ranging from 37-67%. Antibacterial assays revealed that analogues 23-26, in which the (S)-6-methyloctanoic acid moiety is replaced with shorter acyl chains, exhibit distinct antimicrobial activity. The results suggest that the length of the acyl chain is rather critical for antimicrobial activity. On the other hand, substitution of the hydrophobic ring-segment D-Phe-6/Leu-7 in polymyxin B1 with dipeptide mimics (i.e. analogues 27-33) resulted in almost complete loss of antimicrobial activity.     

Localization of cathepsin D in rat liver. Immunocytochemical study using post-embedding immunoenzyme and protein A-gold techniques

Light and electron microscopic localization of cathepsin D in rat liver was investigated by post-embedding immunoenzyme and protein A-gold techniques. By light microscopy, cytoplasmic granules of parenchymal cells and Kupffer cells were stained for cathepsin D. Weak staining was also noted in sinusoidal endothelial cells. In the parenchymal cells many of positive granules located around bile canaliculi. In the Kupffer cells and the endothelial cells, diffuse staining was noted in the cytoplasm in addition to granular staining. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were seen in typical secondary lysosomes and some multivesicular bodies of the parenchymal cells and Kupffer cells. The lysosomes of the endothelial cells and fat-storing cells were weakly labeled. Quantitative analysis of the labeling density in the lysosomes of these three types of cells demonstrated that the lysosomes of parenchymal cells and Kupffer cells are main containers of cathepsin D in rat liver. The results suggest that cathepsin D functions in the intracellular digestive system of parenchymal cells and Kupffer cells but not so much in that of the endothelial cells.     

Changes in trace reactions of sensorimotor cortex and putamen neurons as affected by haloperidol

Experiments on conscious rabbits were made to elaborate motor conditioned reflexes through pairing stimuli with electrocutaneous reinforcement applied every 30 s. Neuronal activity in the sensorimotor cortex and putamen was recorded during formation and reproduction of the conditioned reflexes before and after haloperidol injection (0.2 mg/kg i. v.). In the putamen, haloperidol increased the number of neurons exhibiting trace conditioned activity and made the intensity and duration of these processes rise. The changes seen in the sensorimotor cortex were opposite in nature. Inhibition of trace conditioned activity in the sensorimotor cortex depended mainly on the decreased amplitude of the reaction conditioned component. The role of the dopaminergic system in the interaction of the neostriatum and sensorimotor cortex and in formation and reproduction of trace conditioned activity of both the structures is discussed.