Brefeldin A inhibits oligosaccharide processing of glycoproteins in mouse hypothyroid pituitary tissue at several subcellular sites

We have studied the effects of brefeldin A (BFA) and monensin on the processing of the oligosaccharides of thyrotropin (TSH), free alpha-subunits, and cellular glycoproteins of mouse pituitary tissue to clarify the subcellular sites of action of BFA. BFA was previously shown to inhibit the translocation of glycoproteins from the rough endoplasmic reticulum to the Golgi apparatus but action at other sites was possible. Pituitaries from hypothyroid mice were incubated with [35S]methionine, [3H]mannose, [3H]galactose, [3H]fucose, N-[3H]acetylmannosamine, or [35S]sulfate for 2 hr in the absence or presence of 5 micrograms of BFA/ml or 2 microM monensin. TSH and free alpha-subunits were immunoprecipitated from tissue lysates and analyzed by sodium dodecyl sulfate-gel electrophoresis. The tryptic glycopeptides of TSH were separated using high-performance liquid chromatography. Total glycoproteins in cell lysates were precipitated using trichloroacetic acid. Labeled oligosaccharides were released from the tryptic glycopeptides of TSH and cellular glycoproteins by endoglycosidase H and they were analyzed by paper chromatography. Compared with control incubations, BFA caused the intracellular accumulation of glycoproteins having less than expected amounts of Man9GlcNAc2 units, but with excess Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2 units. There was a lesser accumulation of glucose-containing oligosaccharides, especially Glc1Man9GlcNAc2. Monensin also caused the accumulation of certain high mannose species, but the pattern differed from that seen for BFA, since Man9GlcNAc2 units were preserved and there was less excess of Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2 units. BFA did not block the initial attachment of oligosaccharides at any of the three Asn-glycosylation sites of TSH, but caused the accumulation of Man5-8GlcNAc2 units at each site. Both monensin and BFA inhibited fucosylation, sulfation, and sialylation more markedly than mannose incorporation. Thus, in addition to its previously described action of inhibiting rough endoplasmic reticulum to Golgi transport, BFA appears to partially inhibit the glucose-trimming enzymes as well as some Golgi enzymes.     

Isolation and characterization of streptomycin-resistant mutants in Nicotiana plumbaginifolia

Summary Streptomycin-resistant colonies were isolated from protoplast cultures of haploid Nicotiana plumbaginifolia based on their ability to green in medium containing 1 mg/ml streptomycin sulfate. The frequency of resistant colonies was 0.9×10^–5 in nonmutagenized culture, and increased ten-fold following treatment of culture with 10 g/ml N-methyl-N-nitro-N-nitrosoguanidine. Of a total of 52 resistant clones isolated, 2 gave rise to haploid, 15 to diploid, and 3 to tetraploid plants upon transfer of calli to differentiation medium. Leaf-segment and protoplast assays showed that all diploid regenerates were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, neomycin, and spectinomycin. Plants in most diploid clones were fertile and able to set seeds when self-fertilized and crossed reciprocally to wild-type plants. Inheritance of streptomycin resistance was studied in the diploid clones and, without exception, the resistance was transmitted maternally. Comparative studies of the ultrastructure of organelles and protein synthesis in isolated chloroplasts between wild-type and resistant clones in the presence of streptomycin suggest that streptomycin resistance is controlled by chloroplasts.     

Some physicochemical properties of rice mitochondrial DNA

Summary Certain physicochemical properties of rice mitochondrial DNA (mtDNA) were determined. Certain low-molecular-weight mtDNA bands were found in addition to the major mtDNA band. Rice mtDNA appeared in the electron microscope as a collection of linear molecules with heterogeneous length in the range of 1–156 kb. The major distribution area was 60–105 kb. A small fraction (less than 5%) of rice mtDNA was found in the form of a circular molecule. Some molecules had the appearance of being supercoiled. Replication fork structures were found in both circular and linear mtDNA molecules. In one rice species, Jin Nante, 15 different circular molecules were found. Rice mtDNA was digested with different restriction enzymes. The total molecular weight of rice mtDNA was calculated to be about 300 kb according to the data of restriction enzyme digestion and electron microscopy.     

Anatomical features associated with preaxial duplication (pd): a recessive mutation in the rat

Preaxial polydactyly of the fore- and hindlimbs was found in Wistar-derived rats in 1978. Genetic analysis indicated that the polydactyly was due to the effects of an autosomal recessive gene (gene symbol; pd). Polydactylous homozygous rats had two or three pollices (six or seven digits) in the forelimbs and one to three preaxial extra digits (six to eight digits) in the hindlimbs. Skeletal examination revealed the presence of the extra carpal, metacarpal, and phalangeal bones that seemed to be complete or incomplete duplication of the navicular, greater multangular, first metacarpal, and phalanges of digit I in the forelimbs. In the hindlimbs, extra tarsal, metatarsal, and phalangeal bones were also observed preaxially. These extra elements seemed to be mirror-image duplications of the talus, navicular, second cuneiform, third cuneiform, cuboid, and metatarsals and phalanges of digits II-V with the absence of the first cuneiform, tibiale, first metatarsal, and phalanges of digit I. In addition, morphological changes were observed in the humerus, radius, and ulna in the forelimbs and femur, tibia, and fibula in the hindlimbs. Especially in the radius and tibia, thickening and bifurcation were found, indicating incomplete duplication of these bones. Based on these findings, the limb anomaly was classified as preaxial carpometacarpal/tarsometatarsal-type polydactyly with incomplete duplication of the radius and tibia. The mutant rats had other associated anomalies such as accessory spleens and cryptorchism. The males are sterile, whereas the females breed normally.     

Changes in cerebral level of monoamines by Japanese encephalitis virus infection

The changes in monoamine levels of different brain regions following Japanese encephalitis virus (JEV) intraperitoneal inoculation were examined in experimentally JEV-infected mice. In addition, virus distribution was studied using infectivity assay and immuno-histochemistry of viral antigen. 1) The level of monoamines in brain tissues was not affected by 48 hours after viral inoculation, but marked effects were elicited at 96 hours after the inoculation. The cerebral concentration of 5-hydroxyindole-3-acetic acid (5 HIAA) was increased, while that of dopamine (DA) showed a decrease. Especially these alteration were observed in the cerebral cortex, but not in the cerebellum. 2) The viral growth in the brain was observed at 48 hours after the inoculation. The growth in the cerebellum, however, was found to be lower than those in other cerebral regions. 3) The viral antigen was detected in the cerebral cortex, hippocampus, mesencephalon and diencephalon in addition to the substantia nigra and striatum. From these results, it is presumed that clinical manifestation of JEV infection may involve the changes in the metabolism of neurotransmitter, especially those of DA and serotonin in the brain.     

The same cell lineage is involved in scale formation and regeneration in the teleost fish Hemichromis bimaculatus

The elasmoid scales of the cichlid fish, Hemichromis bimaculatus, are localized within dermal pockets, the floors of which are separated from the stratum compactum by uninterrupted cellular sheets, the scale-pocket linings (SPL). TEM study of the fry skin shows that the SPL cells originate from the cell population constituting the dermal papilla of the scale. The upper-layer cells of the papilla, close to the epidermal-dermal junction, differentiate into scleroblasts that, subsequently, form the scale-bag, while the inner-layer cells, close to the stratum compactum, constitute a bi-layered sheet, the SPL. The SPL cells are joined one to another by numerous desmosomes and their cytoplasm is filled principally by microfilaments and free ribosomes. The SPL is also characterized by the presence of a basement membrane on its two faces. When a scale is experimentally pulled off, the scale-forming cells are removed with the dermis and the epidermis covering the free region of the scale, but the SPL is not damaged and epidermal fragments remain at the posterior edge of each scale-pocket. The epithelial cells migrate, from the epidermal fragments, on an extracellular matrix situated on the surface of the SPL, and the wound is closed from 3 to 6 h after scale removal. The scale-regenerating cells differentiate from the upper-layer cells of the SPL, initially in the central region of the scale-pocket where epithelial cells first contacted the SPL surface. Consequently, it is shown that scale-forming cells and scale-regenerating cells are derived from the same ontogenetic population, the dermal papilla.     

CD16+ lymphoblastic cell lines of crab-eating monkeys (Macaca fascicularis) shared U-5 antigen and expressed natural killer activity

The CD16+ lymphoblastic cell lines of crab-eating monkeys shared the U-5 antigen recognized by a monoclonal antibody. The CD16+U-5+ cell lines expressed high natural killer activity to K562 cells, whereas the CD16-U-5- control cell line had no significant natural killer activity. A possible involvement of the U-5 antigen in natural killer function was also suggested by reduction of the natural killer activity in peripheral blood mononuclear cells of Japanese monkeys after treatment with U-5 monoclonal antibody and complement.     

Development of a series of monoclonal antibodies recognizing leukocyte differentiation antigens of Japanese monkeys (Macaca fuscata)

Six monoclonal antibodies to Japanese monkey leukocytes were developed. These monoclonal antibodies, designated the U series, cover most kinds of leukocytes (pan T cells, CD8+ cells, CD8+ subset and granulocytes, CD16+ cells, monocytes/macrophages), and should be useful in the immunological analysis of primate models, such as tissue transplants and virus-related diseases, in particular, acquired immune deficiency syndrome (AIDS).     

Two structural genes on different chromosomes are required for encoding the major subunit of human red cell glucose-6-phosphate dehydrogenase

Structural analysis revealed the existence of two types of subunits in human red cell glucose-6-phosphate dehydrogenase. The two subunits have the same COOH region consisting of 479 amino acid residues, but their NH2-terminal regions are different in size and sequence. The minor subunit can be fully encoded by the X-linked G6PD cDNA, but the NH2-terminal region of the major subunit cannot. The cDNA and the gene for the NH2-terminal region of the major subunit were cloned and characterized. Southern blot hybridization indicated that the gene for the NH2-terminal region is on chromosome 6, not on the X chromosome. Northern blot hybridization demonstrated an existence of two separate mRNA components, one for the COOH-terminal region and the other for the NH2-terminal region. Two separate structural genes, the X-linked and chromosome 6-linked genes, must be coresponsible for encoding the single chain subunit. Either cross-translation of two mRNAs, or transpeptidation, or some other mechanism must be involved in the synthesis of human red cell G6PD.