A possible role of chorion protease in shell membrane degradation during development of quail embryos.

In the eggs of the quail Coturnix japonica, the limiting membrane demarcates the shell membrane at the interface with the albumen and decreases in width during the hatching process. This study was done to identify agents that affect the width of this limiting membrane. Zymography tests on extracts from extraembryonic tissues, yolk sacs, or chorioallantoic membranes, or all three, showed proteolytic activities during d 4 to 10 of incubation. Localization experiments on these activities, performed on d 5 eggs, indicated that they were located in an avascular chorion. Electron microscopic analysis showed there were secretory cells specifically located in the avascular chorion. After partial purification of d 5 avascular chorion extracts using QA52 and Sephadex G-200 column chromatography, the proteolytic activity of 20 kDa was isolated. The protease showed a high level of activity toward succinyl-Gly-Pro-Leu-Gly-Pro-4-methylcoumaryl-7-amide. It had an optimal pH of 9 and digested the limiting membrane. These enzymatic activities were inhibited moderately by EDTA and strongly by leupeptin and aprotinin. It was concluded that it is the 20-kDa protease, showing collagenase-like activity produced by the avascular chorion, that affects the limiting membrane.

The crystal polymorphism of calcium carbonate is determined by the matrix structure in quail eggs.

Two calcified structures, the eggshell and sperm-associated body (SB), are present in the eggs of the Japanese quail, Coturnix japonica. X-ray diffractometry showed that calcium carbonates take the form of calcite in the eggshell and aragonite in the SB. The aim of the present study was to identify the factors that determine the morphology of calcium carbonate crystals. The matrix of EDTA-treated eggshell was a meshwork of vesicles, 200 to 500 nm in diameter, connected by fine fibers or fibrous sheets. The matrix of SB cortex was a radiation of rod-shaped projections approximately 130 nm in width. In vitro crystal formation was achieved by adding dissociated matrix substances to test solutions. When eggshell matrix material was added, formation of calcite crystals, which had many vesicular holes on their surface, was observed. When SB matrix material dissociated by sonication was added, rhombohedral calcite crystals formed at protein concentrations of 100 microg/mL or lower, and elongated and bundled crystals formed at concentrations of 150 microg/mL or higher. When SB matrix material dissociated by pipetting was added, aragonite crystals formed. These observations indicate that the matrix structure is the principal factor in determining the crystal polymorphism of calcium carbonate.

Early skin toxicity predicts better outcomes, and early tumor shrinkage predicts better response after cetuximab treatment in advanced colorectal cancer.

Cetuximab-containing treatments for metastatic colorectal cancer have been shown to have higher overall response rates and longer progression-free and overall survival than other systemic therapies. Cetuximab-related manifestations, including severe skin toxicity and early tumor shrinkage, have been shown to be predictors of response to cetuximab. We hypothesized that early skin toxicity is a predictor of response and better outcomes in patients with advanced colorectal carcinoma. We retrospectively evaluated 62 patients with colorectal adenocarcinoma who had unresectable tumors and were treated with cetuximab in our institution. Skin toxicity grade was evaluated on each treatment day. Tumor size was evaluated using computed tomography prior to treatment and 4-8 weeks after the start of treatment with cetuximab.Patients with early tumor shrinkage after starting treatment with cetuximab had a significantly higher overall response rate (P = 0.0001). Patients with early skin toxicity showed significantly longer overall survival (P = 0.0305), and patients with higher skin toxicity grades had longer progression-free survival (P = 0.0168).We have shown that early tumor shrinkage, early onset of skin toxicity, and high skin toxicity grade are predictors of treatment efficacy and/or outcome in patients with advanced colorectal carcinoma treated with cetuximab.

Oviducal Contribution to Alteration of the Vitelline Coat in the Frog, Rana japonica. An Electron Microscopic Study.

The vitelline coat (VC) surrounding coelomic eggs of the frog, Rana japonica, comprises bundles of filaments running both parallel and perpendicular to the egg surface. The coat gives little or no staining reaction with PA-CrA-Silver methenamine. In contrast, in the VC of uterine eggs the filament bundles are less conspicuous. and the interstices between the filament bundles stain strongly for carbohydrate. This alteration occurs during passage of the eggs down the first 1/20 th of the oviduct, the pars recta. The epithelium of the p. recta contains secretory cells, which contain electron-dense granules distinct from those in the jelly-secreting cells in more caudal portions of the oviduct. Treatment of coelomic eggs with an extract of p. recta followed by exposure to a sperm suspension resulted in marked swelling and softening of the VC. These results indicate that the contents of the granules secreted from the epithelial cells in the p. recta are deposited in the VC to increase its susceptibility to a fertilizing sperm.

Vitellogenesis-related ovary cathepsin D from Xenopus laevis: purification and properties in comparison with liver cathepsin D.

Cathepsin D was purified from ovaries of Xenopus laevis by both QAE-cellulose and pepstatin-Sepharose chromatography and then characterized and compared with Xenopus liver cathepsin D. Ovary cathepsin D appeared predominantly as a 43-kilodalton (kDa) molecular mass, as revealed by SDS-polyacrylamide gel electrophoresis, whereas the liver enzyme was obtained exclusively as a 36-kDa protein. The purified 43-kDa ovary enzyme cleaved vitellogenin limitedly to produce yolk proteins at pH 5.6. The specific activity of ovary cathepsin D was five to six times lower than that of the liver enzyme, as measured by hemoglobin-hydrolysis at pH 3, but the ovary enzyme was shown to be superior to the liver enzyme in terms of vitellogenin-cleaving activity, as examined at pH 5.6. Ovarian enzyme preparations contained variable amounts of 36-kDa species; this form was considered to be an autolytic product of the 43-kDa form arising during purification, because it was not detected in oocyte extracts but was generated by incubation of the purified 43-kDa enzyme alone in an acid solution. The conversion of the 43-kDa form by hepatic factors was accompanied by a marked increase in hemoglobin-hydrolytic activity.

Purification and properties of embryonic cysteine proteinase which participates in yolk-lysis of Xenopus laevis.

The study reported here aimed to purify a cysteine proteinase from neurula embryos of Xenopus laevis, since this enzyme was thought to be involved in yolk-lysis in developing embryos. The purification procedure consisted of fractionation of an embryonic extract by means of 30-90% ammonium sulfate, chromatography on diethylaminoethyl cellulose and carboxymethyl cellulose, gel filtration on Sephadex G-75 and affinity chromatography on concanavalin A-agarose. The purified enzyme had a molecular weight of 30 kDa according to both SDS-PAGE and Sephadex G-75 gel-filtration and an optimum pH of 5.5, and it preferentially cleaved the synthetic substrate, Z-Phe-Arg-MCA. Its activity was inhibited by Z-Phe-Phe-CHN2, a specific cathepsin L inhibitor, as well as by leupeptin and E-64. The NH2-terminal amino acid sequence of the enzyme was similar to that of chicken cathepsin B. These characteristics indicate that the purified enzyme is a member of the cysteine proteinase family. The antibody raised against the purified enzyme specifically stained a 30 kDa protein of neurula embryo extracts on immunoblot tests. The enzyme effectively digested Xenopus yolk proteins when the NaCl concentration in test solutions was 0.2 M. It was also confirmed that cysteine proteinase inhibitors inhibited yolk-lysis by the enzyme.

Degenerative hairlets on the vestibular sensory cells in mutant bustling (BUS/Idr) mice.

The bustling mouse (BUS/Idr: bus) is a mutant mouse strain which exhibits deafness, bustling/hyperkinetic behaviour and functional disorders seemingly related to the vestibular system. This phenotype develops in homozygous (bus/bus) mice and has been shown from cross experiments to be genetically induced by a single autosomal recessive gene. We previously detected, with light and electron microscopy, post-natal degeneration of the inner ear sensory cells in homozygotes. In the present study, we examined, by electron microscopy, the development of pathological changes in the sensory epithelia of the macula acustica and crista ampullaris of homozygous mice of various ages, paying special attention to the detailed morphology of the sensory hairlets. The homozygous mice exhibited specific pathological changes: a decrease in the number of hairs; disarrangement of the kinocilium-stereocilia pattern; and, fused and/or very large stereocilia. Homozygotes also frequently exhibited apical cytoplasmic herniation, or bleb of hair cells, as well as a degenerated kinocilium in the sensory epithelium. Heterozygotes showed similar changes, but to a lesser degree and frequency. As for the vestibular organs, similar pathological changes had developed at day, 17 of gestation. These pathological findings and onset suggest that the BUS mouse may be a mutant mouse strain distinct from other reported strains which display similar behaviour, and may be a useful animal model for the study of human degenerative vestibular disorders.

Changes in shell membranes during the development of quail embryos.

The shell membrane of an avian egg acts as a bag enclosing albumen and water. At its interface with the albumen, a smooth layer of homogeneous, dense material called the limiting membrane demarcates the shell membrane. The present study aimed to investigate changes in the limiting membrane during development of quail embryos that were grown with or without being turned. Sixty-three percent of the embryos were hatched after the eggs were incubated at 39 C and in 60% humidity with automatic rotation around their long axis and with their equatorial side down, whereas the hatch rate decreased to 24% when the eggs were incubated without being turned. The width of the limiting membrane at the equatorial region of turned eggs gradually decreased from 74 nm on Days 0 to 2 of incubation to 35 nm on Day 10 and thereafter. Conversely, water permeability, measured by evaporation through the shell membrane increased from 4 to 5 nL/mm2 per min on Days 0 to 6, to 9 nL/mm2 per min on Day 12 and thereafter. In stationary eggs, the decrease in the width of the limiting membrane on the lower side of eggs was delayed until Day 8 of incubation. The water permeability of the shell membrane in this group was 51% of that of the membrane on the upper side of eggs on Day 8 of incubation. Forty to forty-four nanometers seemed to be the critical width of the limiting membrane at which high water permeation could occur. It was also shown that the albumen hinders water permeation through the membrane. These results show that (1) the limiting membrane is made thin during the development over the whole surface with egg-turning, possibly through digestion of still unknown agents, and (2) this thinning accelerates the rate of water permeation through the membrane.

[A case of chronic myelogenous leukemia complicated with nephrotic syndrome].

An 81-year-old woman was admitted, complained general malaise, and edema on face and lower extremities. In the peripheral blood, leucocytosis (17,220/mm3), microcytic hypochromic anemia (RBC 348 x 10(4)/mm3, Hb 9.6 g/dl, Ht 29.2%), and thrombocytosis (130 x 10(4)/mm3) were present, and many myeloid cells containing of myeloblasts, promyelocytes and so on were observed. Bone marrow aspiration revealed increment of the myeloid series without hiatus leukemia . The Neutrophil Alkaline Phosphatase score and rate was low, and on bone marrow scintigram using indium chloride, liver and extremities were shown. On admission, proteinuria (21.5 g/dl) and hypoalbuminemia (2.5 g/day) were pointed out, and the renal biopsy specimen showed membraneous proliferative glomerulonephritis (MPGN), so we diagnosed this case that chronic myelogenous leukemia (CML) complicated with nephrotic syndrome. At first, she was treated with prednisolone, but proteinuria was not entirely improved, then busulfan was given, myeloid cells in peripheral blood were disappeared and proteinuria was gradually decreased. From this coarse, the causality between CML and nephrotic syndrome was verified.

Ionic induction of the frog cement-gland cell from presumptive ectodermal tissues.

Cells of the superficial layer which had been explanted from the presumptive ectoderm of Rana japonica early gastrulae at stage 10 differentiated into cement-gland cells (CGCs) when cultured in Barth's solution containing 90-130 mM-NaCl, and into common epidermal cells and cilia cells when cultured in a solution containing 20-40 mM-NaCl. They failed to differentiate, however, when cultured in a solution in which NaCl is 15 mM or lower. The optimum condition for inducing the differentiation of CGC was stimulating them with a solution containing 130 mM-NaCl for 6-10 h at 18 degrees C, followed by culturing in a solution containing 15-40 mM-NaCl for 7 days. The greatest ability to react to the CGC-inducing stimuli resided in the superficial layer of the presumptive ectoderm of the embryo at stages 10-11. Under the optimum condition, the total volume of CGCs induced amounted to about 85% of the explanted tissue. High percentage comparable to this was obtained with stimulation by KCl, RbCl, sucrose or mannitol.

Cysteine proteinase plays a key role for the initiation of yolk digestion during development of Xenopus laevis.

In electrophoretic analyses, extracts of Xenopus laevis neurulae exhibited activities digesting yolk proteins maximally at pH 4.8. These activities were completely inhibited by a mixture of pepstatin A and Z-Phe-Phe-CHN2, thus being identifiable as cathepsin D and cysteine proteinase. The electrophoretic profiles of yolk proteins cleaved by embryonic extracts changed at gastrula stages; the profile before stage 13 was the same as that given by cathepsin D treatment and the profile at stage 13 was a combination of the profile given by cathepsin D treatment and that given by cysteine proteinase treatment. Quantitative measurement of enzyme activities showed that the cathepsin D activity that was preserved from the beginning of development increased from stages 13 to 25 and decreased thereafter, whereas the cysteine proteinase activity appeared at stage 13, gradually increased until stage 35 and strongly increased thereafter. Immunoblot analyses showed that the 43 kDa form of cathepsin D was processed to its 36 kDa form, presumably by cysteine proteinase. This change can explain the increase of cathepsin D activity at stage 13 and thereafter. Immunofluorescent staining with the antibody against cysteine proteinase occurred in mesodermal and ectodermal cells other than neural ones at stages 13-24, and in the endodermal cells at stages 24-36. Faint staining in the neural ectoderm persisted from stages 18 to 36. Immunoelectron microscope observation showed that what stained was the superficial layer of yolk platelets. All these results indicate that cysteine proteinase plays a key role in the initiation of yolk digestion during embryonic development.

Changes in the chorion and sperm entry into the micropyle during fertilization in the teleostean fish, Oryzias latipes.

Specific antibodies against the major chorionic glycoproteins (ZI1-2 and ZI3) of unfertilized eggs were used to analyze the differences in the chorion and its surrounding constituents before and after fertilization. The glycoproteins in the inner layers of the chorion and its surrounding material were specifically stained by both of the antibodies. Thirty and 60 min after activation, the thickness of the chorion's inner layers was already reduced and the micropylar canal was closed. At the same time, the broadly diluted mucous area (DMA) of glycoproteins on the outermost layer of the chorion in unfertilized eggs was modified to a thin, compact layer. When unfertilized eggs were treated with trypsin, the inner third portion of the micropylar canal closed and the glycoproteins in the DMA were digested. The incidence of sperm entry into the micropyle of these eggs was extremely reduced. These results suggest that in medaka eggs, the chorionic glycoproteins in the DMA on the chorion surface, which have an affinity for spermatozoa, play an important role in sperm guidance into the micropyle.

Analyses of oviductal pars recta-induced fertilizability of coelomic eggs in Xenopus laevis.

The acquisition of fertilizability in coelomic eggs of Xenopus laevis has been shown to be correlated with the physical, biochemical, and ultrastructural alterations of the egg envelope [coelomic envelope (CE)] induced during the passage of eggs through the pars recta portion of the oviduct. However, no direct evidence that the pars recta renders eggs fertilizable has yet been presented. In this study, we show that coelomic eggs are highly fertilizable when they are incubated with continuous shaking for 4 h at 15 degrees C in pars recta extract (PRE) derived from females prestimulated by pregnant mare serum gonadotropin. The PRE from pituitary-stimulated Bufo japonicus was as potent as homologous PRE in rendering Xenopus eggs fertilizable. Incubation of coelomic eggs in PRE for 30 min induced a dramatic increase in the rates of sperm binding to the envelope to a level equivalent to that exhibited by the envelope from uterine eggs (VEs). The CE-to-VE ultrastructural conversion and a 43k-to-41k hydrolysis of the envelope glycoprotein component started 5 min after, and were completed by 15 min after, the start of incubation in PRE and were accompanied by an exposure of a new N-terminal sequence typical to gp41. Thus, the biochemical and ultrastructural conversions and the sperm-binding activity of the envelope induced by PREs, although being prerequisite, were not sufficient to render coelomic eggs fully accessible to fertilizing sperm.

Augmented expression of a type IV collagen-binding protein in a highly metastatic murine fibrosarcoma clone.

The adhesive properties of highly and weakly metastatic murine sarcoma (Meth A) clones were investigated. A highly metastatic clone, MH-02, preferentially adhered to type IV collagen-coated plastic dishes and to bovine pulmonary arterial endothelial cell-coated plastic dishes as compared to a weakly metastatic clone, ML-01. Pretreatment of MH-02 and ML-01 cells with antisera against MH-02 cells resulted in almost equivalent adhesiveness to type IV collagen. Preincubation of 125I-radiolabeled tumor cells with the antisera against MH-02 significantly reduced the arrest of MH-02 cells in the lung, but ML-01 cells were not affected. The number of pulmonary metastatic nodules of MH-02 cells was reduced to the same level as that of ML-01 cells by preincubation of the tumor cells with the antisera in an experimental metastasis experiment. These results indicated that the high metastatic ability of MH-02 can be attributed to its preferential adhesiveness to type IV collagen. The type IV collagen-binding proteins of MH-02 and ML-01 were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Among several proteins which bound to type IV collagen, expression of a protein with a molecular weight of 29 kD was significantly greater in MH-02 than in ML-01. These results suggest that the greater adhesion of highly metastatic MH-02 cells to type IV collagen is due to enhanced expression of the type IV collagen-binding 29 kD protein.

Externalization of tropomyosin isoform 5 in colon epithelial cells.

Ulcerative colitis (UC) is associated with autoantibody response to a cytoskeletal protein, human tropomyosin (hTM) isoform-5 (hTM5). Because hTM5 is an intracellular protein, it may remain inaccessible to the autoantibodies. Therefore, we have investigated the possibility of externalization of hTM5 in colon epithelial cells. Freshly isolated colonic and small intestinal epithelial cells and LS-180 colon cancer cell line were examined for surface expression of hTM5 by flow cytometric analysis using hTM isoform-specific MoAbs. The extracellular release of hTM5 was determined by Western blot and radioimmunoprecipitation analyses. Physical association of hTM5 with a membrane-associated colon epithelial protein (CEP) was examined by co-immunoprecipitation of hTM5 with anti-CEP MoAb, and CEP with anti-hTM5 MoAb. Cell surface expression of hTM5 was observed in colonic epithelial and LS-180 cells but not in small intestinal epithelial cells. LS-180 cells spontaneously released hTM5 as well as CEP into the culture medium that was significantly stimulated by a calcium ionophore, A23187, but inhibited by phorbol-12-myristate-13-acetate, monensin and methylamine. Co-immunoprecipitation experiments revealed that hTM5 forms a complex with CEP. We conclude that hTM5 is externalized in colon but not in small intestinal epithelial cells. The physical association of hTM5 with CEP suggests a possible chaperone function of CEP in the transport of hTM5, a putative target autoantigen in UC.

The peri-albumen layer: a novel structure in the envelopes of an avian egg.

The present paper describes a novel structure, termed the peri-albumen layer, in the egg-envelopes of the quail Coturnix japonica. It reacts with Alcian blue and exists between the egg white and the shell membrane. Ultrastructurally, it is of fine granular structure and forms a fenestrate sheet, the width of which is 190 nm or less. Isolated materials of the peri-albumen layer include an Alcian-blue-positive polysaccharide of 260 kDa, and three glycoproteins of 160, 108 and 52 kDa. The layer is supplied to an egg when it passes through the magnum-isthmus junction, the normalized length of which is 0.62-0.63 of the oviduct. The mucosa of the junction consists exclusively of a luminal epithelium. It is apparently distinct from the mucosa of the magnum and the isthmus, which consist of a luminal epithelium and tubular glands. The luminal epithelium of the magnum-isthmus junction stains prominently with Alcian blue and consists of alternately distributed ciliated cells and granular cells. Immunohistochemistry with an antiserum raised against the materials of the peri-albumen layer revealed the staining of the peri-albumen layer of the egg, and secretory cells of the luminal epithelium at the magnum-isthmus junction. It was concluded that the materials of the peri-albumen layer are produced by secretory cells at the magnum-isthmus junction of the oviduct.

Over 80% of NotI sites are associated with CpG rich islands in the sequenced human DNA.

Clones containing DNA segments linked to NotI sites are not only useful for ordering the NotI fragments fractionated by pulsed field gel, but also valuable in the search of unknown genes, because they often contain the CpG rich islands and genes related with them. To know the probability of association of NotI sites with CpG rich islands, we screened 5,188 sequences accumulated in DNA data base for the presence of NotI site and examined the distribution of CpGs around them. The sequential calculation of G + C content and frequency of CpG occurrence at each nucleotide position identified the CpG rich domains close to NotI sites in 77 sequences, which corresponds to 84% of total number of candidate sequences. This frequency is consistent well with the prediction that 89% of NotI sites in mammalian genome are likely to be present in CpG rich islands and would stress the importance of cloning of NotI linking sequences for direct isolation of desired genes. Furthermore, 63 islands newly identified in this study should provide a clue for understanding the transcriptional regulation of associated genes.