Research Note: Expression of IGF-1 and IGF-1 receptor proteins in skeletal muscle fiber types in chickens with hepatic fibrosis.

We investigated the expression of insulin-like growth factor 1 (IGF-1) and IGF-1 type 1 receptor (IGF-1R) in skeletal muscle fiber types in chickens with hepatic fibrosis induced by bile duct ligation (BDL). Eleven hens, approximately 104 weeks old, were randomly assigned to BDL (n = 4) and sham surgery (SHAM; n = 7) groups. In BDL hens, histopathology revealed marked bile duct proliferation and liver fibrosis. The cross-sectional area (CSA) of myofibers from both the pectoralis (PCT) muscles significantly decreased in the BDL group compared with the SHAM group (P < 0.01). In contrast, the CSA of myofibers from the femorotibialis lateralis (FTL) muscle did not decrease in the BDL group. Type I fibers were large, round, and hypertrophic. Elongated type IIA and IIB fibers were also present. For IGF-1 immunostaining, the immunoreaction intensity was higher in the PCT in the BDL group than the SHAM group. Within the BDL group, type I fibers from FTL had a stronger immunoreaction intensity than the type II fibers. For IGF-1R immunostaining, the intensity of the immunoreactions was similar within the PCT in the BDL group compared with the SHAM group. For FTL, type I fibers had stronger reactions to IGF-1R than type II fibers in the BDL group. These results suggest that type I fibers express both IGF-1 and IGF-1R and become hypertrophic in chickens with hepatic fibrosis.

The Rho guanine nucleotide exchange factor ARHGEF5 promotes tumor malignancy via epithelial-mesenchymal transition.

Epithelial tumor cells often acquire malignant properties, such as invasion/metastasis and uncontrolled cell growth, by undergoing epithelial-mesenchymal transition (EMT). However, the mechanisms by which EMT contributes to malignant progression remain elusive. Here we show that the Rho guanine nucleotide exchange factor (GEF) ARHGEF5 promotes tumor malignancy in a manner dependent on EMT status. We previously identified ARHGEF5, a member of the Dbl family of GEFs, as a multifunctional mediator of Src-induced cell invasion and tumor growth. In the present study, ARHGEF5 was upregulated during tumor growth factor-ß-induced EMT in human epithelial MCF10A cells, and promoted cell migration by activating the Rho-ROCK pathway. ARHGEF5 was necessary for the invasive and in vivo metastatic activity of human colorectal cancer HCT116 cells. These findings underscore the crucial role of ARHGEF5 in cell migration and invasion/metastasis. An in vivo tumorigenesis assay revealed that ARHGEF5 had the potential to promote tumor growth via the phosphatidylinositol 3-kinase (PI3K) pathway. However, ARHGEF5 was not required for tumor growth in epithelial-like human colorectal cancer HCT116 and HT29 cells, whereas the growth of mesenchymal-like SW480 and SW620 cells depended on ARHGEF5. Induction of EMT by tumor necrosis factor-α or Slug in HCT116 cells resulted in the dependence of tumor growth on ARHGEF5. In these mesenchymal-like cells, Akt was activated via ARHGEF5 and its activity was required for tumor growth. Analysis of a transcriptome data set revealed that the combination of ARHGEF5 upregulation and E-cadherin downregulation or Snail upregulation was significantly correlated with poor prognosis in patients with colorectal cancers. Taken together, our findings suggest that EMT-induced ARHGEF5 activation contributes to the progression of tumor malignancy. ARHGEF5 may serve as a potential therapeutic target in a subset of malignant tumors that have undergone EMT.

Deregulated expression of a novel component of TFTC/STAGA histone acetyltransferase complexes, rat SGF29, in hepatocellular carcinoma: possible implication for the oncogenic potential of c-Myc.

c-Myc N-terminal conserved domains, MbI and MbII, are essential for c-Myc-mediated transformation and transactivation. These domains recruit the STAGA (SPT3-TAF9-GCN5-acetyltransferase) coactivator complex, but not TFTC (TATA-binding protein-free TAF-containing) to the target gene promoter. Although components of this complex are well conserved between yeast and mammals, four mammalian orthologs of yeast SPT8, SPT20, SGF11 and SGF29 remain to be identified. Here, we isolated a rat ortholog of yeast SGF29, a component of yeast SAGA (SPT-ADA-GCN5-acetyltransferase) complex. Both rat (r) SGF29 and c-myc mRNAs were overexpressed in five out of the eight tested rodent tumor cells. rSGF29 directly interacted with rADA3 and co-immunoprecipitated with two other TFTC/STAGA components, rGCN5 and rSPT3. rSGF29 was recruited to the c-Myc target gene promoters together with c-Myc, and it activated c-Myc target gene expressions. Downregulation of rSGF29 suppressed the expression of c-Myc target genes and inhibited anchorage-independent growth and tumorigenicity and lung metastasis of rat hepatoma K2 cells when injected into nude mice. These results show that rSGF29 is a novel component of TFTC/STAGA complexes and could be involved in the c-Myc-mediated malignant transformation.

Movement of endoplasmic reticulum in the living axon is distinct from other membranous vesicles in its rate, form, and sensitivity to microtubule inhibitors.

The endoplasmic reticulum (ER) is the major membranous component present throughout the axon. Although other membranous structures such as synaptic vesicles are known to move via fast axonal transport, the dynamics of ER in the axon still remains unknown. To study the dynamics of ER in the axon, we have directly visualized the movement of two ER-specific membrane proteins, the sarcoplasmic/endoplasmic reticulum calcium-ATPase and the inositol 1,4,5-trisphosphate receptor, both of which were tagged with green fluorescence protein (GFP) and expressed in cultured chick dorsal root ganglion neurons. In contrast to GFP-tagged synaptophysin that moved as vesicles at 1 microm/sec predominantly in the anterograde direction in the typical style of fast axonal transport, the two ER proteins did not move in a discrete vesicular form. Their movement determined by the fluorescence recovery after photobleaching technique was bi-directional, 10-fold slower (approximately 0.1 microm/sec), and temperature-sensitive. The rate of movement of ER was also sensitive to low doses of vinblastine and nocodazole that did not affect the rate of synaptophysin-GFP, further suggesting that it is also distinct from the well-documented movement of membranous vesicles in its relation with microtubules.

Morphological changes and recovery process in the tenotomized soleus muscles of the rat.

Tenotomized soleus muscles of adult rats were analyzed morphologically and biochemically with special reference to the recovery process. Light microscopic observations of semi-thin sections showed that the characteristic central core lesion was most extensive at 1 week after tenotomy and began to diminish in extent at 2 weeks until no trace of lesion could be seen by 6th week, as confirmed by thin-section electron microscopy. Three phases of changes in the cross-sectional area of muscle fibers after tenotomy were demonstrated by morphometry: phase I designated as the initial increase up to the 3rd day, phase II as the progressive decrease until the 4th week, and phase III as the recovery to normal or even hypertrophy. In electron microscopy, the earliest alteration of myofibrils was recognized at 3 days after tenotomy. The Z discs showed a wavy or zigzag profile with frequent longitudinal splitting of myofibrils. From the 2nd week on, muscle fibers underwent a process of recovery, replacing the central core lesion with new myofibrils in which a reassembly of thick filaments into bundles of thin filaments took place, with Z discs being aligned adjacent to the peripheral complete myofibrils. In SDS-polyacrylamide gel electrophoresis, the molar ratio of myosin to actin diminished markedly as the central core lesion developed and gradually returned to normal with time, correlating well with the loss and subsequent reassembly of thick filaments.

Increased solubility of high-molecular-mass neurofilament subunit by suppression of dephosphorylation: its relation to axonal transport.

To investigate the role of phosphorylation in the turnover and transport of neurofilament (NF) proteins in vivo, we studied their solubility properties and axonal transport in the rat sciatic nerve using phosphatase inhibitors to minimize dephosphorylation during preparation. About 20% of the 200-kDa subunit (NF-H) in the axon was soluble in the 1% Triton-containing buffer under the present conditions, whereas this amount was less and more variable in the absence of phosphatase inhibitors. The 68-kDa subunit (NF-L) was exclusively insoluble and not affected by the inhibitors. Such selective solubilization of NF-H by phosphorylation differed significantly from the in vitro phosphorylation with cyclic AMP-dependent protein kinase, which resulted in NF disassembly. The carboxy-terminal phosphorylation state of NF-H probed with the phosphorylation-sensitive antibodies was also not directly related to solubility. The solubility of NF-H did not differ along the nerve. In contrast, the solubility of L-[35S]methionine-labeled, transported NF-H was lowest at the peak of radioactivity. Higher solubility at the leading edge, regardless of its location along the nerve, indicates that NF-H solubility is positively correlated with the rate of NF transport.

Immunohistochemical study of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 human intervertebral discs.

STUDY DESIGN: Immunohistologic staining of human intervertebral discs collected at the time of surgery (100 intervertebral discs from 80 patients) and 10 discs collected from 7 cadavers within 12 hours of death was performed using antimatrix metalloproteinase-3 monoclonal antibody and antitissue inhibitor of metalloproteinase-1 monoclonal antibody. OBJECTIVES: To examine the relationship between matrix destruction and staining for matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 in intervertebral disc degeneration. SUMMARY OF BACKGROUND DATA: Matrix metalloproteinase-3, which decomposes aggregating proteoglycans, has attracted research attention as a substance contributing to matrix destruction in the articular cartilage and intervertebral disc. However, except for a few in vitro studies, the relationship between matrix destruction of the intervertebral disc and matrix metalloproteinase-3 has been little studied. METHODS: Immunohistologic staining was performed to examine the relationship between matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 in the intervertebral disc, and the relationship of these two agents to magnetic resonance imaging, radiographic, and surgical findings. RESULTS: Those cases testing positive for matrix metalloproteinase-3 and negative for tissue inhibitor of metalloproteinase-1 accounted for most of the surgical specimens. The matrix metalloproteinase-3-positive cell ratio was significantly correlated with the magnetic resonance imaging grade of intervertebral disc degeneration, and the matrix metalloproteinase-3-positive cell ratio observed in prolapsed lumbar intervertebral discs was significantly higher than that in nonprolapsed discs. In cervical intervertebral discs, the matrix metalloproteinase-3-positive cell ratio and staining of cartilaginous endplate were correlated with the size of osteophyte formation. CONCLUSIONS: These findings suggested that intervertebral disc degeneration is caused by disturbance in the equilibrium of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1, and that matrix metalloproteinase-3 contributes to degeneration of the cartilaginous endplate.

Phosphorylation and transport of neurofilament proteins in the rat spinal ganglion.

Neurofilament proteins (NFs) in rat spinal ganglia were labeled with [32P]orthophosphate injected into ganglia and analyzed by two-dimensional autoradiography and immunoblotting. Three polypeptides of NF were labeled irrespective of the extent of phosphorylation. Most of the labeled NFs were transported from cell bodies to proximal axons within 24 h. A major fraction of low phosphorylated NF-H changed to high phosphorylated form in intraganglionic nerve fibers and peripheral nerves adjacent to spinal ganglia. A small fraction of low phosphorylated NF-H appeared earlier than the high phosphorylated form in adjacent peripheral nerves, suggesting that newly synthesized NF-H in cell bodies migrate a long distance before they are extensively phosphorylated and assembled into the cytoskeleton in proximal axons.

Biochemical characterization of nerve growth cones isolated from both fetal and neonatal rat forebrains: the growth cone particle fraction mainly consists of axonal growth cones in both stages.

Nerve growth cones are responsible for the exact pathway finding, and for the establishment of neurocytoarchitecture. To elucidate the developmental changes of biochemical characteristics of nerve growth cones, growth cone particle (GCP) fractions were isolated biochemically from embryonal day 17 (E17) rat forebrain and from postnatal day 5 (P5). There were no significant differences in protein phosphorylation pattern in a Ca(2+)-dependent manner between E17-GCP fraction and that of P5. As for the membrane lipid composition, molar ratios of cholesterol to total phospholipids were well conserved during these ages. The immunoreactivity to anti-synaptophysin monoclonal antibody as a marker of mature synaptic elements could not be detected either in E17-GCP or P5-GCP fractions. To exclude the possibility of the contamination of dendritic elements, RNA contents and immunoreactivity to anti-high molecular weight microtubule-associated protein 2 (MAP2) monoclonal antibody were examined. RNA contents of the GCP fractions were extremely low compared to those of other subcellular fractions both in E17 and P5. No immunoreactivities to anti-MAP2 antibody were observed in either GCP fraction. Our results suggest that the GCP fractions, isolated from forebrains of E17 to P5 rat, are free from the contamination of the synaptic elements, and that the GCP fractions are mainly composed of axonal growth cones.

[Three patients with typical sandblaster's silicosis proven by mineralogical analysis].

Three family members who had worked as sandblasters in their own sandblasting factory showed innumerable small nodularities in both lung fields of their chest radiograms. One of those showed conglomerate shadows in the upper lung fields. Those shadows seemed to be consistent with those of silicosis. One of the patients was examined by transbronchial lung biopsy (TBLB) and showed typical silicotic nodules. Mineralogical studies were done on the abrasive particles and the deposited particles in the lung tissue specimen obtained via TBLB and bronchoalveolar lavage fluid sample (BALF) using polarized microscopy, X-ray diffraction and analytical electron microscopy. The particles which had accumulated on the floor of the factory mineralogically consisted of mostly (over 90%) silica quartz containing small amount of chlorite, and the deposited particles in the lung tissue and those in the BALF showed similar composition.

Effects of taxol on slow and fast axonal transport.

Axonal transport of tubulin in the rat sciatic nerve is almost completely inhibited by a single subepineural injection of taxol, without affecting that of neurofilament proteins. Actin and a large number of polypeptides cotransported with actin as minor components are also blocked by taxol, although to a lesser extent. Fast axonal transport is essentially free from the inhibitory effect of this drug. Although previous models have suggested that slow axonal transport involves the bulk movement of cytoskeletal structures, these results suggest that such transport may involve an equilibrium between polymerised and depolymerised forms of the axonal cytoskeleton.

Two populations of axonally transported tubulin differentiated by their interactions with neurofilaments.

In the sensory fibers of the rat sciatic nerve (fibers of the dorsal root ganglion cells), two components of tubulin transport were observed that differed in the rate of transport, solubility in Triton, and subunit composition. The faster component, migrating ahead of the neurofilament proteins, was soluble in 1% Triton. The slower component, migrating with the neurofilament proteins, was insoluble in 1% Triton and contained a unique polypeptide, "NAP," in the tubulin region that was not present in the faster component. "NAP" was not a subspecies of tubulin as evidenced by peptide mapping. It seems to be a neurofilament-associated protein. When a complete separation of the main tubulin wave from the neurofilament wave was achieved in the motor axons of the same nerve (axons of the ventral motoneurons) under the effect of beta,beta'-iminodipropionitrile, a portion of tubulin was still found associated with the retarded neurofilament wave. The subunit composition of this portion was similar to the slower, neurofilament-associated component in the sensory fibers under normal conditions, i.e., enriched in "NAP" and the most acidic subtype of beta-tubulin. It is suggested that two populations of transported tubulin exist that are differentiated by the extent of their interaction with neurofilaments.

Subunit composition specific to axonally transported tubulin.

One week after injection of L-[35S]methionine into the dorsal motor nuclei of the guinea-pig, labelled tubulin carried down the vagal nerve by the slow phase of axonal transport was analysed by one- and two-dimensional gel electrophoresis. Transported tubulin showed a much stronger labelling of the beta-subunit. Isoelectric focussing revealed that both alpha- and beta-subunits were composed of several components. Labelled tubulin was isolated from the brain by cycles of polymerisation and depolymerisation after injection of L-[35S]methionine into the lateral ventricle, for comparison with transported tubulin from the vagal nerve. In addition to the two alpha-components and three beta-components detected in both preparations, axonally transported tubulin contained an extra component (TAX) with a molecular weight corresponding to that of beta-tubulin and with the same isoelectric point as alpha-tubulin. The axon-specific component TAX co-polymerised with tubulin isolated from the brain. Upon peptide mapping by limited proteolysis, the peptide pattern generated from TAX was similar to that of the alpha-tubulin. It is concluded that the axonally transported tubulin contains a modified alpha-subunit which is not found in the bulk of brain tubulin.

[Combination chemotherapy with cis-diammine-di-chloro-platinum (II) and bleomycin in advanced head and neck cancer].

Combination chemotherapy of cis-diammine dichloro platinum (II) (CDDP) and bleomycin was given to 10 patients with advanced squamous cell carcinoma of the head and neck. Nine patients had received prior radical radiotherapy, 2 had major ablative surgical procedures, and one had been previously treated with chemotherapy. Responses were as follows (duration in months): 2 CRs (4,6+), 2 PRs (1.5,1.5), and 2 minors. Vomiting related to CDDP was observed in 5 patients, nephrotoxicity and hypocalcemia in one patient were also observed.

Preferential blockade of the tubulin transport by colchicine.

L-[35S]Methionine was injected into the dorsal root ganglion (L5) of the adult rat, and migration of the neurofilament polypeptides (the triplet with molecular weights of 200,000, 160,000 and 68,000 daltons), alpha- and beta-tubulins and actin in the sciatic nerve and the dorsal root was quantitatively determined and also examined by fluorography. Colchicine (4 microgram) injected into the ganglion 10 min before methionine preferentially blocked the tubulin transport, with little if any blockade of the triplet and actin. Colchicine at this dose had no effects on the incorporation of L-[14C]leucine into the total protein and also into tubulins. In contrast to colchicine, vinblastine sulphate (4 microgram) injected into the ganglion in a similar way blocked the transport of all the triplet, tubulins and actin. Cytochalasin D (1 microgram) had no effect on the slow axoplasmic transport.