Sequential polymerization of flagellin A and flagellin B into Caulobacter flagella

The mode of polymerization of two species of flagellins, flagellin A and flagellin B, in polar flagella of Caulobacter crescentus was examined. By immunological staining we found that 1 to 1.2 μm of the portion of the flagellar filament proximal to the cell was composed of flagellin B, whereas about 5 μm of the distal portion was composed of flagellin A. This result, together with the previous observation that a flagellin B-less mutant cannot form normal flagella but instead forms stubs in spite of their high level of flagellin A synthesis, indicates that flagellin B is very important for the formation of complete flagella and/or for the initiation of filament formation from the hook.     

Effects of Hydrostatic Pressure on Carcinogenic Properties of Epithelia

The relationship between chronic inflammation and cancer is well known. The inflammation increases the permeability of blood vessels and consequently elevates pressure in the interstitial tissues. However, there have been only a few reports on the effects of hydrostatic pressure on cultured cells, and the relationship between elevated hydrostatic pressure and cell properties related to malignant tumors is less well understood. Therefore, we investigated the effects of hydrostatic pressure on the cultured epithelial cells seeded on permeable filters. Surprisingly, hydrostatic pressure from basal to apical side induced epithelial stratification in Madin-Darby canine kidney (MDCK) I and Caco-2 cells, and cavities with microvilli and tight junctions around their surfaces were formed within the multi-layered epithelia. The hydrostatic pressure gradient also promoted cell proliferation, suppressed cell apoptosis, and increased transepithelial ion permeability. The inhibition of protein kinase A (PKA) promoted epithelial stratification by the hydrostatic pressure whereas the activation of PKA led to suppressed epithelial stratification. These results indicate the role of the hydrostatic pressure gradient in the regulation of various epithelial cell functions. The findings in this study may provide clues for the development of a novel strategy for the treatment of the carcinoma.     

A monoclonal antibody to the carbohydrate chain on human hepatocellular carcinoma-associated antigen which suppressed tumor growth in nude mice

Summary There have been few reports stating that monoclonal antibody alone inhibits human solid tumor growth in vivo. The present study demonstrated that monoclonal antibody S1 (IgG2a), which recognized the antigenic determinant of the carbohydrate moiety, showed antibody-dependent cell (or macrophage)-mediated cytotoxicity (ADCC or ADMC) in conjunction with murine splenocytes of both BALB/c and athymic mice. In vivo experiments demonstrated that the antibody S1 clearly prolonged the survival of athymic mice which had been inoculated with a human liver carcinoma cell line. In addition, the antibody S1 significantly suppressed the human hepatoma line transplanted s.c. into nude mice. ¹²⁵I-Labeled monoclonal antibody S1 revealed that the antibody accumulated significantly in the tumor mass. Many mononuclear cells were observed surrounding tumor cells when the antibody was given. This model system might be useful for analyzing the ADCC (or ADMC) mechanism in vivo.     

Comparative Study of Glial Marker Proteins in the Hypoplastic Cerebellum of Jaundiced Gunn Rats

The behavior of marker proteins of glial cells [alpha-enolase, beta-S100 protein, and glial fibrillary acidic protein (GFAP)] was investigated quantitatively by using enzyme immunoassay systems during the development of cerebellar hypoplasia in jaundiced Gunn rats. A neuronal marker protein, gamma-enolase, was also measured as a reference. At postnatal day 8 corresponding to the early stage of cerebellar damage, the amount of beta-S100 on a protein basis was significantly higher in jaundiced homozygotes (jj) than in control nonjaundiced heterozygotes (j+), whereas no differences in alpha- and gamma-enolases and GFAP were observed between the two groups of rats. At days 15 and 30, which correspond, respectively, to the advanced and late stages of cerebellar damage, the three glial proteins, especially GFAP, were higher and the neuronal protein was lower in the jj rat cerebellum than in the control. These results are consistent with the reported histological observations that neuronal cells are vulnerable and damaged by bilirubin, whereas glial cells seem to be less sensitive. On the other hand, the amounts of beta-S100 and alpha-enolase per cerebellum were significantly lower in jj rats at days 15 and 30, as in the case of gamma-enolase, whereas that of GFAP remained at the same level as the control at day 15 and showed a slight but significant decrease at day 30. The possibility is suggested that beta-S100 and GFAP may be available as biochemical indicators of glial cells, especially in the early and advanced stages of cerebellar damage, respectively, but that alpha-enolase is less available.     

Effect of thyrotropin releasing hormone injection on blood growth hormone (GH), TSH and growth hormone releasing hormone (GHRH) concentrations in cancer patients

In order to investigate whether endogenous GHRH and somatostatin were involved in the mechanism of the paradoxical GH rise after TRH injection, changes in serum GH and plasma GHRH were examined before and after TRH injection in 12 cancer patients and changes in serum TSH and GH were similarly studied in 76 cancer patients including 31 GH-responders and 45 GH-nonresponders to TRH. TRH stimulated GH secretions without altering the circulating GHRH concentration in 4 of the 12 cancer patients. There was neither a significant correlation between the increase from the basal to maximum GH and GHRH after TRH injection in the 12 cancer patients nor a reciprocal relationship between the increase in GH and TSH after TRH injection in the 76 cancer patients. These findings suggested that the paradoxical GH rise after TRH injection in cancer patients was exerted by its direct action at the pituitary level, and not mediated through the hypothalamus.     

Immunological analysis of glycolipids on cell surfaces of cultured human tumor cell lines: expression of lactoneotetraosylceramide on tumor cell surfaces

Glycolipids of human cell lines of colonic adenocarcinoma (Colo 205 and BM 314), gastric tumor (AZ 521 and KATO-III), and lung tumor (A 549) were studied by the immunohistochemical fluorescence technique, flow cytometric analysis and immunostaining on thin layer chromatoplates with antibodies against gangliotriaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), fucogangliotetraosylceramide (Fuc-Gg4Cer), blood group B active lipid, globopentaosylceramide (Gb5Cer) and lactoneotetraosylceramide (nLc4Cer). Anti-nLc4Cer antibody was the only antibody which reacted with all the tumor cell lines used. The glycolipid fractions of each cell line separated by Iatrobeads column chromatography were immunostained with the six antibodies mentioned above on thin layer plates. The presence of nLc4Cer was detected in all cell lines. On the other hand, Gg4Cer was detected in gastric tumor cell lines, and Gg3Cer was detected in AZ 521. Based on these results, the tumor cell lines were analyzed by flow cytometry using anti-nLc4Cer antibody. About 70% of total cells in each cell line were separated as nLc4Cer-expressing cells. The present findings, together with the occurrence of nLc4Cer in ascitic fluids of cancer patients (Taki, T., Kojima, S., Seto, H., Yamada, H., & Matsumoto, M. (1984) J. Biochem. 96, 1257-1265), suggest that nLc4Cer may be a tumor-associated lipid.     

Immunologic characterization and molecular profile of carcinoembryonic antigen detected by monoclonal antibodies

Four distinct monoclonal antibodies, which reacted with CEA preparations but not with nonspecific cross-reacting antigen or with nonspecific cross-reacting antigen 2, were established. Except for monoclonal antibody AS001 , all of these monoclonal antibodies immunoprecipitated molecular forms of 200K and 180K daltons that are not bridged by disulfide bonds. Immunodepletion experiments and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that these monoclonal antibodies recognized the same antigenic structure when 125I-CEA preparation was used. Monoclonal antibody AS001 is of particular interest, because this antibody reacted only with a 200K dalton molecule which is a part of the molecules recognized by the other three monoclonal antibodies. The rosette inhibition assay and the immunoprecipitation experiments suggest that each monoclonal antibody recognizes a different antigenic determinant. The antigenic determinants recognized by monoclonal antibodies YK013 and AS001 may be peptides in nature, whereas the determinants recognized by antibodies YK024 or AS005 might be carbohydrate. The radioimmunoassay with monoclonal antibody AS001 was established, and the results clearly indicate that the incidence of positivity for the sera from digestive tract cancer patients and from lung cancer patients obtained by monoclonal antibody AS001 was higher than that obtained by the polyclonal antibody. Monoclonal antibody AS001 was able to detect the corresponding antigen in the sera, which the polyclonal antibody failed to detect. This study therefore suggests that monoclonal antibodies may enhance and improve the diagnostic value in cancer patients with undetectable or lower CEA levels detected by conventional anti-CEA antibodies.     

Sex difference in the paradoxical response of serum GH to thyrotrophin releasing hormone in cancer patients

It was demonstrated that thyrotropin-releasing hormone (TRH) elicited a paradoxical increase in basal GH levels in cancer patients. Out of 94 cancer patients, 50 were found to be GH responders and this phenomenon was more frequently recognized in female than in male cancer patients. In cancer patients under 59 years of age, the GH response to TRH was significantly greater in females than in males, although there was no sex difference in the GH response in patients above 60 years of age. In female cancer patients, the GH response to TRH was significantly greater in patients under 59 years of age than in patients above 60 years of age, while there was no age difference in the GH response in male cancer patients. It was concluded that paradoxical responses of serum GH to TRH were recognized in 53 per cent of cancer patients and were more frequently observed in female than in male cancer patients.     

Wound healing in the cornea of the chick embryo

Spatio-temporal changes in the shapes of the epithelial cells in culture were followed with the aid of scanning electron microscopy. On a substratum that enables the epithelium to spread extensively, the first remarkable change in shapes of the cells occurred at the margin of epithelium at 12 h of culture. The marginal cells formed leading edges with filo- or lamellipodia, flattened, and lost microvilli on surface. In accordance with those changes, the borderlines among cells became almost indiscrenible. Flattening of the cells was the essential characteristic associated with active epithelial spreading throughout the culture period. Elongation of cells of intermediate zone at right angles to the direction of the locomotion of the marginal cells at 24 h of culture was the second significant change. As the third, the change from the ordinary pentagonal or hexagonal to extraordinary tetragonal or other polygonal shapes, with or without irregular margins, began in cells of the intermediate area at 24 h and propagated to those in inner area. The active deformation of the inner cells with no space in which to move was considered to play some role in the extensive epithelial spreading.