Sterol regulatory element-binding proteins (SREBPs) regulate transcription of genes encoding enzymes in the cholesterol biosynthetic pathway and the LDL receptor. These proteins are synthesized as membrane-bound precursors and processed to generate the NH₂-terminal domains, mature transcription factors. We established two Chinese hamster ovary (CHO) cell lines, CHO-421 expressing the truncated hamster SREBP-1 (amino acids 421-1133) with two transmenbrane domains and CHO-557 expressing another truncated SREBP-1 (amino acids 557-1133) without any transmembrane domains, to investigate the fate of the COOH terminus after cleavage of the NH₂-terminal mature SREBP. The cell fractionation experiments revealed that the two proteins, regardless of the absence of transmembrane domains in the SREBP (557-1133), similarly localized in the nuclear envelope and the microsomal membrane fractions, suggesting that these proteins appear to be tightly bound to a membrane protein(s) localizing on the nuclear and endoplasmic reticulum (ER) membranes. Although we predicted that overexpression of the COOH-terminal domains, which were thought to be involved in the regulation of SREBP processing, would result in disruption of the SREBP-dependent transcriptional regulation of several genes, the mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase in these two cell lines were regulated in a sterol-dependent manner. Pulse-chase experiments revealed that the SREBP (421-1133) was relatively rapidly degraded (t_1/2=4-6 hr) and that the intracellular cholesterol level did not affect the half-life time. The degradation of the SREBP (421-1133) was not suppressed by the treatment of a calpain inhibitor, N-acetyl-leucyl-leucyl-norleucynal (ALLN), which blocks the proteolysis of some proteins within or near the ER. In CHO-557 cells the SREBP (557-1133) was much more rapidly degraded (t_1/2=1-2 hr), suggesting that the cytosolic COOH-terminal domain is accessible to the enzymatic attacks from the cytoplasm. Taken together, overexpression of the COOH-terminal domains does not affect the regulation of SREBP processing and the domains are rapidly turned over by the cytosolic proteolytic process distinct from the ALLN-sensitive ER degradative pathway.

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Vitronectin is a cell-adhesive glycoprotein in blood plasma and extracellular matrix. We have examined the participation of vitronectin in experimental metastasis of B16F10 mouse melanoma cells which metastasized to lung. By Western blotting, a protein reacted with anti-rat vitronectin antibody was found to be enriched more in the melanoma parenchyma in the lung than the sum of cultured melanoma cells and normal lung tissue. The protein was similarly abundant in tumors of B16F10 cells grown subcutaneously in mice. By immunofluorescence of the tissues, the signal detected with anti-rat vitronectin was found to be localized at nuclei in melanoma cells metastasized in the lung as well as in tumors just grown subcutaneously. These results indicate that the protein reacted with anti-vitronectin antibody is expressed much more in the growing mass of B16F10 melanoma cells in vivo than in vitro.

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Low affinity nerve growth factor receptor (p75^LNGFR) it is thought to play an important role in recovering damaged nerve. To investigate the possible role of p75^LNGFR in transient retinal ischemia, we investigated p75^LNGFR gene expression and localization.
Using rats under anesthetized conditions, we incised the bulbar conjunctive around the limb us, and then clamped the eyes. A sham operation was performed on the contralateral eyes. Ocular ischemia was maintained for 90 minutes. The p75^LNGFR gene expression in ischemic rat retinas was examined by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) at 0, 3, 6, and 72 hours after reperfusion, and the localization of p75^LNGFR protein in rat retinas was examined by light and electron microscopic immunohistochemistry.
The expression of p75^LNGFR gene in ischemic rat retinas increased, as compared with that of the contralateral eyes after 6 hours and 3 days of reperfusion. The p75^LNGFR protein increased in the outer plexiform layer and in the outer limiting membrane by immunohistochemical technique. Electron microscopic immunohistochemistry demonstrated that the staining is present in the Müller glial cells.
The fact that p75^LNGFR gene expression increased in Müller cells after reperfusion suggested that p75^LNGFR expression may play a curative role in ischemic injury.

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Microtubule-associated proteins (MAPs) promote the assembly of microtubules from purified tubulin in vitro. In order to establish a model system for the investigation of the role of MAPs in micro tubule assembly in vivo, we have generated Saccharomyces cerevisiae strains that permit the modulation of the expression levels of MHP1 (MAP-Homologous Protein 1) and of the α and β-tiibulin genes. Simultaneous overexpression of α and β tubulin results in the accumulation of long aberrant microtubules in interphase, a similar phenotype as was observed in cells overexpressing MHP1. We demonstrate that overexpression of MHP1 in asynchronously growing yeast cultures leads to cell cycle arrest in G2. In cells that overexpress MHP1 and the tubulin genes, a suppression of both the MHP1 and the tubulin overexpression phenotypes can be observed. Progressive induction of α and β tubulin overexpression and constitutive overexpression of MHP1 lead to the formation of long cytoplasmic microtubules more frequently than observed in cells overproducing tubulin or Mhplp individually and the increased microtubule polymerization could be correlated with the increase of α and β tubulin expression. However, the overexpression of MHP1 did not alter the phenotypes of individual overexpression of α or β-tubulin. These data indicate that Mhplp not only stabilizes microtubules but promotes microtubule assembly in vivo, and suggest that the role of other mammalian MAPs in the promotion of microtubule assembly could be tested in this yeast system.

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The exposure of freshly isolated osteoblasts and osteoblast-like cells to high-level hypergravity caused the inhibition of cell growth, elevation of cAMP content, and the stimulation of differentiated functions such as alkaline phosphatase activity, collagen synthesis, and osteocalcin synthesis. Blockage of elevation of cAMP by SQ22536, an inhibitor of adenylate cyclase, resulted in the inhibition of the hypergravity-stimulated alkaline phosphatase activity, indicating that cAMP is the intracellular mediator of this action of hypergravity. H89, an inhibitor of cAMP-dependent protein kinase (PKA), further inhibited the cell growth that was already inhibited by the hypergravity, and further stimulated the alkaline phosphatase activity that was already stimulated by hypergravity. If cAMP acts through the PKA system, H89 should have blocked the changes in cell function effected by the exposure to hypergravity. Therefore the elevated intracellular cAMP by the exposure of hypergravity caused the changes in cell function by a PKA-independent pathway.

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It is well known that DNA-ploidy is useful independent prognosticator of malignancy. However, the biological significance of polyploid cells and the relation between polyploidy and prognosis is not well understood. We analyzed DNA ploidy by flow cytometry in Meth-A cells (a cultured sarcoma cell line) after treatment with K252a, a protein kinase inhibitor, and showed induction of polyploidization. Apoptotic cell death of the high polyploid cells was verified by flow cytometry, morphological observation and gel analysis of DNA integrity. Expression of tumor-suppressor nuclear protein p53 investigated by immunohistochemistry was increased 10-fold or more in cells with 16C (C = haploid DNA content) relative to cells with 2C, suggesting that the overexpression of p53 was involved in the apoptosis. These results may be of clinical relevance since it has been known that both DNA ploidy and p53 expression have prognostic significance.

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