Identification of lamin B2 as a substrate of protein kinase C in BALB/MK-2 mouse keratinocytes

Protein phosphorylation by activation of protein kinase C was examined using quiescent cultures of the mouse epidermal keratinocyte line BALB/MK-2. Treatment with phorbol ester caused rapid phosphorylation of five proteins with molecular weights of 80,000, 70,000, 40,000, 34,000, 28,000. Of these proteins, the 70,000 molecular weight one (p70) was studied further. Its position on two-dimensional gel suggested that p70 is nuclear envelope lamin B. This possibility was confirmed by the co-migration of p70 with the lamin fraction of mouse liver and its immunoprecipitation with antinuclear lamina antibody. The lamin B fraction consists of lamin B1 and lamin B2. Evidence that p70 is lamin B2 was obtained by peptide mapping and amino acid sequencing. Lamin B2 is the only lamin that shows a substantial increase in phosphorylation on treatment of BALB/MK-2 cells with phorbol ester.     

Breakpoint Junction of Interstitial Homozygous Deletion at Chromosome 2q33 in a Small Cell Lung Carcinoma

We have characterized the breakpoint junction of the homozygousdeletion at chromosome 2q33 in a small cell lung carcinoma cellline. Cloning and sequencing of the genomic regions surroundingthe breakpoint junction of the deletion revealed that the homozygousdeletion was caused by a simple interstitial deletion of a 220-kbsegment. An AT-dinucleotide of contributing germline sequenceswas overlapped at the junction. Since there were one or twonucleotide overlaps of germline sequences at breakpoint junctionsin all four cases of interstitial deletions analyzed to date,this may reflect a common mechanism underlying the occurrenceof chromosomal interstitial deletion.     

Radiation-induced aneusomic clones in bone marrow of rats.

Wistar rats 3 months old were given a single whole-body X-irradiation with 700 R. They were killed 9.3 months, on average, after irradiation. From the bone marrows of the 23 irradiated rats, 54 clones of cells with radiation-induced chromosome abnormalities, ranging from 3.3 to 78.3% in size, were obtained. Karyotype analysis at the banding level showed that 43 out of the 54 clones had balanced chromosome constitutions, and that the remaining 11 clones were unbalanced. The 43 balanced clones consisted of 33 clones with reciprocal translocations, 6 with inversions and 4 with both translocations and inversions. The 11 unbalanced clones were made up of 7 aneuploid clones and 4 pseudo-diploid clones. Of the 54 clones, 15 were large with frequencies of more than 25%. Contrary to general belief that cells with unbalanced chromosome constitutions have less capacity to proliferate than those with balanced ones, 8 of the 15 large clones, especially all, except 1, of the largest 6 clones were unbalanced, either aneuploid or pseudo-diploid.     

Tumor-promoting, phorbol ester-induced phosphorylation of cell-surface transferrin receptors in human erythroleukemia cells

When human erythroleukemia cells (K562) were exposed to phorbol-12-myristate 13-acetate (PMA), phosphorylation of transferrin receptors was enhanced 5-fold with 10(-7) M PMA and 7-fold with 10(-6) M PMA, but not with 4 alpha-phorbol (5 X 10(-7) M). Stimulation took place in serine residues in the cytoplasmic domain of the receptor. Although phosphorylation in the control cells took place in both cell-surface and intracellular receptors, phosphorylation in PMA-treated cells increased only in the cell-surface receptors, not in the intracellular receptors. The number of receptors on the cell surface increased slightly with the increase in phosphorylation at the cell surface, in the PMA-treated cells. No difference in transferrin binding was found for the control and PMA-treated cells. These results indicate that enhanced phosphorylation of the transferrin receptor takes place on the cell surface only and that it presumably is mediated by protein kinase C.     

Lipid droplets containing DNA-histones released in the culture medium of transformed rat fibroblasts (HY1)

Lipid droplets appeared during the growing phase in the culture medium of incompletely transformed rat fibroblasts (HY1) induced by AccI-H fragments of adenovirus 12 DNA. These droplets consisted of neutral lipids, DNA, histones and RNA. Electron microscopic observations showed that the droplets had no lipid-bilayers on their surfaces which accounted for the tendency of the droplets to readily fuse together and become larger, and that the inner structures of the droplets looked like networks of fibrous matter. Experiments with DNA hybridization showed that the droplet DNA was composed of both cellular and adenoviral DNA, and that the cellular DNA in the droplets seemed to be derived from various cell DNA sequences. These results suggest that the droplets were derived from parts of the nuclear components of HY1 cells. The mechanisms for droplet release are discussed.     

Intra- and extracellular localization of hyaluronic acid and proteoglycan constituents (chondroitin sulfate, keratan sulfate, and protein core) in articular cartilage of rabbit tibia.

To demonstrate the intra- and extracellular localization of hyaluronic acid (HA) in articular cartilage of the rabbit tibia, biotinylated HA binding region, which specifically binds to the HA molecule, was applied to the tissue. In comparison with the localization of HA, that of chondroitin sulfate (CS), keratan sulfate (KS), and the protein core (PC) of the proteoglycan was examined by immunohistochemistry. Strong positive staining for HA was detected in chondrocytes located in the transition between the superficial and middle zones of the tissue. Pre-treatment with chondroitinase ABC, keratanase II, or trypsin enhanced the stainability for HA in peri- and intercellular matrices. Immunohistochemistry with or without enzymatic pre-treatment demonstrated that immunoreactivity for CS, KS, and PC was distinctly discerned in chondrocytes and in the extracellular matrix located in the middle and deep zones. In particular, the immunoreactivity for KS and PC was augmented by pre-treatment with chondroitinase ABC not only in chondrocytes but in the extracellular matrix located in the middle and deep zones. Microbiochemical analysis corresponded well with histochemical and immunohistochemical results. These results suggest that HA is abundantly synthesized and secreted in chondrocytes located in the transition between the superficial and middle zones.     

Fish assemblage structure response to seagrass bed degradation due to overgrazing by the green sea turtle Chelonia mydas at Iriomote Island,southern Japan

Ichthyological Research - The fish assemblage structure response to rapid degradation of Enhalus acoroides seagrass beds due to overgrazing by green sea turtles (Chelonia mydas) was investigated at...