Regulation of the expression of vimentin gene during the differentiation of mouse myeloid leukemia cells.

We have examined the expression of vimentin during the differentiation of mouse myeloid leukemia cells (M1), which were induced to differentiate into macrophages by exposure to conditioned medium (CM) obtained from rat embryo fibroblasts. The synthesis of vimentin, which was examined by two-dimensional gel electrophoresis, increased after 12-24 h of incubation of M1 cells in CM and the elevated level of synthesis continued up to 96 h. A macrophage cell line (Mm1) that was derived from spontaneously differentiated M1 cells constantly synthesized much higher levels of vimentin. The amount of vimentin, which was revealed by immunoblot analysis using an mAb against human vimentin, also increased after differentiation by a factor of 7 when compared on the basis of constant protein and by a factor of 17 on the basis of constant cell numbers. Mm1 cells contained greater than 12- and 45-fold more vimentin compared with undifferentiated M1 cells on the bases of constant protein and constant cell numbers, respectively. Northern blot analysis using vimentin cDNA as a probe revealed increases in vimentin mRNA in the differentiated M1 cells and Mm1 cells. Nuclear run-on assay showed that the expression of vimentin gene during the differentiation of M1 cells was transcriptionally regulated. Observations in indirect immunofluorescence microscopy and EM clearly showed that vimentin bundles were rarely observed in undifferentiated M1 cells, and increased amounts of and large-size vimentin bundles were easily observed in differentiated M1 and Mm1 cells. These results suggest the participation of increased amounts of vimentin filaments in the maldistribution of nuclei in M1 cells during differentiation.

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) activates the maternal program of apoptosis shortly after MBT in Xenopus embryos.

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) mRNA in 1- and 2-cell stage Xenopus embryos induces cell autonomous dissociation at the late blastula stage and developmental arrest at the early gastrula stage. The induction of cell dissociation took place "punctually" at the late blastula stage in the SAMDC-overexpressing cells, irrespective of the stage of the microinjection of SAMDC mRNA. When we examined the cells undergoing the dissociation, we found that they were TUNEL-positive and contained fragmented nuclei with condensed chromatin and fragmented DNA. Furthermore, by injecting Xenopus Bcl-2 mRNA together with SAMDC mRNA, we showed that SAMDC-overexpressing embryos are rescued completely by Bcl-2 and becometadpoles. These results indicatethat cell dissociation induced by SAMDC overexpression is due to apoptotic cell death. Since the level of S-adenosylmethionine (SAM) is greatly reduced in SAMDC-overexpressing embryos and this induces inhibition of protein synthesis accompanied by the inhibition of DNA and RNA syntheses, we conclude that deficiency in SAM induced by SAMDC overexpression activates the maternal program of apoptosis in Xenopus embryos at the late blastula stage, but not before. We propose that this mechanism serves as a surveillance mechanism to check and eliminate cells physiologically damaged during the cleavage stage.

Modulation of cell surface lectin receptors on K562 human erythroleukemia cells induced by transfection with annexin IV cDNA.

Annexin IV was found to be highly expressed in various human adenocarcinoma cell lines, but not in an erythroleukemia cell line, K562. We investigated the effects of transfection of human annexin IV cDNA into K562 cells on cell surface lectin receptors. cDNA transfectants were found to be more sensitive to cytotoxic lectins such as Ricinus communis agglutinin and wheat germ agglutinin than mock transfectants. The results of flow cytometric analyses with various lectins showed that the transfectants expressed more sugar chains which bind to Ulex europaeus agglutinin I and Maackia amurensis mitogen than mock transfectants. These results suggest that transfection of annexin IV cDNA increases the expression of alpha-2,3-sialylated and/or fucosylated sugar chains on the surface.

Inhibition of glucocorticoid-induced apoptosis by the expression of antisense gene of mitochondrial ATPase subunit 6(1).

To isolate the apoptosis-linked genes involved in the cell death of thymocytes induced by glucocorticoids, we developed a functional cloning assay. Murine CD4(+)CD8(+) thymic cell line 2-257-20 cells were transfected with cDNA expression libraries obtained from a dexamethasone-resistant cell line. The transfected cells were selected in the presence of dexamethasone, and the plasmids which episomally expanded were then extracted from the surviving cells. One of the rescued cDNAs was found to be an antisense cDNA fragment identical to the mouse mitochondrial ATPase 6 gene. In the stable transfectants with the ATPase 6 antisense gene, the induction of apoptosis by dexamethasone was significantly delayed. Furthermore, the ATP synthesis in these transfectants was also reduced to some extent. ATPase 6 is a subunit of F(o)F(1) ATPase and our results support that ATP synthesis from the mitochondria is necessary for the induction of apoptosis induced by glucocorticoids.

Assessment of procedures and instruments for intracordal injection: evaluation of the syringe for periodontal ligament anesthesia (CITO JECT).

The intracordal injection technique was first introduced by Brünings in 1911. This technique has been accepted as vocal rehabilitation for dysphonia caused by deficiencies of glottal closure. Although various injection materials have been evaluated, the injection procedure itself has not been studied. In this study, we measured the mechanical force required to perform intracordal injection by using a certain amount of substance, when varying the needle size and syringe type. Although a large needle can reduce the mechanical force for injection, leakage from the pinhole can not be prevented. We modified this technique by introducing a CITO JECT, a syringe, which is used for the anesthesia of the periodontal ligament, to perform the collagen injection. With this technique, the mechanical force required for the injection can be reduced to 1/2 to 1/4 of the force by the conventional technique. We conclude that the introduction of this syringe enable us to use the needle with smaller diameter to facilitate the intracordal injection.

Expression of carbohydrate-binding protein p33/41 in human tumor cell lines.

We previously reported a new type of lectin, p33/41 (annexin IV), which was isolated from a bovine tissue extract [Kojima, K. et al. (1992) J. Biol. Chem. 267, 20536-20539]. When the expression of p33/41 (annexin IV) was surveyed in the lysates of 39 human tumor cell lines by SDS-PAGE, followed by Western blot analysis with polyclonal anti-bovine p33/41 and monoclonal anti-annexin IV (Z016, Zymed) antibodies, 21 cell lines were found to be reactive with the polyclonal antibody, whereas all 39 cell lines were stained with Z016. These results together with those obtained with standard proteins, annexins IV and V, suggested that the monoclonal antibody, Z016, recognizes annexin V, but not p33/41 (annexin IV). Therefore, we performed cDNA cloning of human p33/41 (annexin IV) to prepare a recombinant protein and raised monoclonal antibodies against the protein. Northern blot analysis with the cDNA as a probe showed that a human colon cancer cell line, HT29, contains p33/41 (annexin IV) mRNA of two sizes, 2.0 and 3.0 kb. The two monoclonal antibodies, AS11 and AS17, against the recombinant protein generated were useful for flow cytometric analysis, ELISA, Western blot analysis and immunoprecipitation. Flow cytometric analysis with AS17 showed that p33/41 (annexin IV) is located in the cytoplasm of HT29 cells, but not on the cell surface. However, one of the cell surface proteins first labeled with biotin and then solubilized with a detergent was immunoprecipitated with AS17. The results suggest the existence of a membrane spanning form of p33/41 (annexin IV).

Relation of abdominal and thigh adipose tissue distribution to serum lipids and glucose metabolism in obese males.

Spin-echo magnetic resonance imaging (MRI) and postprocessing for fat quantification were used to examine the relationship of abdominal and thigh adipose-tissue distribution to serum lipids and glucose metabolism in obesity. Thirteen simple obese male patients and 12 non-obese male volunteers were examined by MRI, blood pressure, and fasting blood sample levels of serum lipids, glucose, immunoreactive insulin, c-peptide, HbA1C and hematocrit. Correlations of thigh visceral and subcutaneous fat areas to serum lipid levels were generally similar, but marked differences were found between relationships of thigh versus abdominal fat areas to serum lipid levels. In addition, diastolic blood pressure was significantly correlated with the fat area, especially with the abdominal visceral fat area (r=0.51, p<0.01), but not with abdominal subcutaneous fat area. The thigh muscle area was highly and inversely correlated with c-peptide (r=-0.72, p<0.01) and systolic blood pressure (r=-0.65). Differences in correlations between visceral and subcutaneous fat areas in the abdomen to metabolic parameters were found between abdominal visceral fat areas and HbA1C and between the abdominal subcutaneous fat areas and HbA1C. These findings suggest that the character of regional fat could be heterogeneous with respect to lipid and glucose metabolism and blood pressure levels in obese males.

Maternal program of apoptosis activated shortly after midblastula transition by overexpression of S-adenosylmethionine decarboxylase in Xenopus early embryos.

When we studied polyamine metabolism in Xenopus embryos, we cloned the cDNA for Xenopus S-adenosylmethionine decarboxylase (SAMDC), which converts SAM (S-adenosylmethionine), the methyl donor, into decarboxylated SAM (dcSAM), the aminopropyl donor, and microinjected its in vitro transcribed mRNA into Xenopus fertilized eggs. We found here that the mRNA injection induces a SAM deficient state in early embryos due to over-function of the overexpressed SAMDC, which in turn induces inhibition of protein synthesis. Such embryos developed quite normally until blastula stage, but stopped development at the early gastrula stage, due to induction of massive cell dissociation and cell autolysis, irrespective of the dosage and stage of the mRNA injection. We found that the dissociated cells were TUNEL-positive, contained fragmented nuclei with ladder-forming DNA, and furthermore, rescued completely by coinjection of Bcl-2 mRNA. Thus, overexpression of SAMDC in Xenopus embryos appeared to switch on apoptotic program, probably via inhibition of protein synthesis. Here, we briefly review our results together with those reported from other laboratories. After discussing the general importance of this newly discovered apoptotic program, we propose that the maternal program of apoptosis serves as a surveillance mechanism to eliminate metabolically severely-damaged cells and functions as a 'fail-safe' mechanism for normal development in Xenopus embryos.

Expression of carbonic anhydrase I or II and correlation to clinical aspects of colorectal cancer.

BACKGROUND/AIMS: This study was undertaken to elucidate the correlation between the expression of carbonic anhydrase I or II and the characteristic features of colorectal cancer. METHODOLOGY: The carbonic anhydrase I or II expressions of 74 colorectal cancer patients were analyzed by Western blotting. The relative intensity of cancer to the paired normal mucosa was calculated, and then compared with the clinicopathological parameters. Furthermore, a multivariate analysis for synchronous distant metastasis was undertaken. RESULTS: The expression of carbonic anhydrase I in colon cancer or carbonic anhydrase II in rectal cancer with Duke's D was found to be significantly lower than that with Duke's B or C, respectively. Similarly, carbonic anhydrase I in colon cancer or carbonic anhydrase II in rectal cancer with moderate-severe budding was found to be significantly lower than that with none-mild budding, respectively. Based on the findings of a logistic regression analysis, carbonic anhydrase I was adopted for colon cancer (P = 0.057) and carbonic anhydrase II for rectal cancer (P = 0.008) regarding synchronous distant metastasis. CONCLUSIONS: The expressions of carbonic anhydrase I and II correlated with biological aggressiveness of colorectal cancer and synchronous distant metastasis, especially carbonic anhydrase I for colon cancer and carbonic anhydrase II for rectal cancer.

Imaging techniques for measuring adipose-tissue distribution in the abdomen: a comparison between computed tomography and 1.5-tesla magnetic resonance spin-echo imaging.

Eight subjects were examined both by abdominal X-ray computed transverse axial tomography (CT) and magnetic resonance imaging (MRI) (SE) (TR/TE, 200 ms/15 ms); another eight volunteers were subjected to three MRI scans to test the reliability of repeated measures. Correlations between fat area measures obtained by CT and by MRI for subcutaneous fat, total fat, and visceral vs. subcutaneous-fat ratio were highly significant (r = 0.93, 0.91, and 0.94, respectively; p < 0.01), and the standard errors of estimation were 9.99, 23.87, and 0.0047. The average errors of the method for different fat areas were 2.20 cm2 (intra-examination variance) and 3.75 cm2 (inter-examination variance) for visceral and 0.82 cm2 (intra-examination variance) and 1.29 cm2 (inter-examination variance) for subcutaneous fat areas, respectively. These results suggest that SE MRI is a practical approach to evaluate body fat distribution without the exposure to radiation. The reproducibility of SE MRI for the determination of fat areas is high; variation is small and acceptable. However, it is difficult to determine which estimate of fat area should be accepted when there is a discrepancy between MRI and CT measures.

Is injectable collagen truly safe?

Most patients have no response to injectable collagen or silicone, but some cases may have positive or 'undersea' (= clinically negative but immunologically positive) response to collagen. From the results of the Macrophage migration inhibition test, the relative immunogenicity was augmented most when we used implants with the following combination. The first immunization was collagen and the second one was collagen with silicone. The augmented antigenicity might be enough to cause an allergic reaction to the patients who had no response to each implant alone.

Inhibition of head and neck metastatic and/or recurrent cancer by local administration of multi-cytokine inducer OK-432.

The multi-cytokine inducer OK-432 is a pulverized preparation of the low-virulence SU strain of Streptococcus pyogenes of human origin. A reduction of the tumour mass in the OK-432-injected areas was observed in 11 out of 13 patients with metastatic and/or recurrent head and neck cancer. Complete response (CR), partial response (PR) and minor response (MR) were noted in six, three and two cases respectively. OK-432 local administration therapy could create a new strategy for cancer therapy.

[Indication and effectiveness of endoscopic percutaneous gastrostomy as a route of parenteral alimentation for the home care patient].

We are managing 8 home care patients who have a gastrostomy made using an endoscopic percutaneous technique as a route of parenteral alimentation. Based on our experience, the preconditions for an endoscopic percutaneous gastrostomy as a route of parenteral alimentation are 1. normal gastrointestinal function, 2. difficulty in swallowing, 3. possibility that the caregiver can manage the gastrostomy. When we performed an endoscopic percutaneous gastrostomy as a route of parenteral alimentation for 8 home care patients, we obtained the several advantages mentioned below. 1. Swallowing pneumonia was prevented. 2. Adequate amount of alimental liquid could be infused. 3. Patient could take a bath or shower with the gastrostomy, and good QOL was realized. 4. The home care patient with the gastrostomy could have a satisfactorily long life.

Analysis of the susceptibility of CD57 T cells to CD3-mediated apoptosis.

After stimulation with anti-CD3 antibody in vitro, CD57(+) T cells showed a greater susceptibility to apoptosis than CD57(-)alphabetaT cell receptor (TCR)(+) T cells (regular alphabeta T cells). The apoptotic fraction of CD57(+) T cells showed an increased production of active caspase-3. An increase in both Fas expression and Fas-ligand (FasL) production was also observed in CD57(+) T cells, whereas the expression of survivin was suppressed in CD57(+) T cells compared to that of regular alphabeta T cells. CD57(+) T cells display a biased expansion of a few Vbeta T cell fractions in individuals, but such Vbeta T cells were not specifically susceptible to CD3-mediated apoptosis. The TCR expression level of CD57(+) T cells was much lower than that of regular T cells and anti-TCR antibody stimulation induced a smaller apoptotic proportion of CD57(+) T cells than did anti-CD3 antibody. Although the CD3epsilon expression levels were similar in both T cell subsets, the CD3zeta level of CD57(+) T cells was significantly higher than that of regular T cells. These results suggest that several apoptotic and anti-apoptotic molecules are involved in the CD3-induced apoptosis of CD57(+) T cells and raise the possibility that the imbalance in expression of the CD3epsilon and CD3zeta chains may also contribute to the susceptibility of CD57(+) T cells to undergo apoptosis.

Purification of AV-1, a position-specific molecule, and the effects of its antiserum on chick limb pattern formation.

AV-1 protein is a membrane-localized glycoprotein which is expressed in a stage- and position-specific manner during limb development and is a candidate pattern formation molecule. In this study, we have purified the AV-1 protein using an affinity column coupled with the anti-AV-1 monoclonal antibody. Using the purified protein, we produced a rat anti-AV-1 antiserum. Characterization of the antiserum by immunoblotting and immunostaining showed highly specific reactivity which was identical to that of the anti-AV-1 monoclonal antibody. The rat anti-AV-1 antiserum was injected into the vitelline vein of stage 20 chick embryos and these embryos were allowed to develop further to examine the effect of the antibody on limb pattern. The injected embryos developed deformed limbs. Many of the abnormalities in limb cartilage pattern were related to the normal pattern of the AV-1 expression, suggesting an involvement of the AV-1 protein in the process of limb pattern formation.

Color pattern formation on the wing of the butterfly Pieris rapae. 1. Cautery induced alteration of scale color and delay of arrangement formation.

Experimental approaches to color pattern formation of lepidopteran insects have been made exclusively by analyzing pattern alterations in adult wings induced by operations. We microcauterized the presumptive black region of the dorsal forewing of the butterfly Pieris rapae and analyzed not only the resultant color pattern in the adult wing but also the cell behavior in the pupal wing epidermis around the injury. Cautery induced color alterations were as follows: (i) cautery up to 49.5 h after pupation resulted in white regions appearing within the black region while later cauteries induced larger white regions; (ii) cautery between 50 and 59.5 h resulted in the white regions induced by the cauteries being dramatically decreased; (iii) cautery after 60 h resulted in white regions that had almost disappeared. The examination of the cell behavior in the pupal wing epidermis after cauteries showed that the row formation of scale precursor cells was delayed. This delayed area varied with the time of cautery, in the same manner as that in the induced white area in the adult wing ((i)-(iii) above). The relationship between scale color alteration and the developmental delay of the scale row formation is discussed.

Color pattern formation on the wing of a butterfly Pieris rapae. 2. Color determination and scale development.

It has been shown that microcautery on the prospective apical black region of the early pupal forewing of a butterfly, Pieris rapae, causes alteration of the scale color on the adult wing and a delay in histogenesis of the pupal wing. From these results, it has been assumed that the developmental delay of scale cells in the pupal wing alters their developmental fate and the hypothesis that different color fates of scales are determined by differences in the developmental timetables between scale cells is proposed. In this study, we attempted to find the developmental timetables of individual scales expressing specific color to test this hypothesis. It was found that the holes on the upper surface of a scale become larger as they develop and the hole sizes of scales in the white region are always larger than in the black region on the same wings either during pupal period or after eclosion. This suggests that the scale hole size is a good index that reflects developmental rate of the scale and a difference in the hole size between adult scales is attributed to a difference in the developmental timetables when their ancestral scale precursor cells were in the pupal period. A comparison of the hole sizes between adult scales in different color regions suggested that normal white scales were in a more advanced state than were the black ones but white scales induced by microcautery were in a less advanced state than black ones on the same wing. This supports our hypothesis.

Enhancement of activation-induced cell death by fibronectin in murine CD4+ CD8+ thymocytes.

Development of T cells in the thymus is achieved through the interactions of thymocytes with their microenvironments. This study focused on the function of fibronectin (FN), a major extracellular matrix molecule in the thymus, in the cell death induced by activation via the T-cell antigen receptor. FN alone did not increase cell death in murine thymocytes above the baseline level, but it significantly enhanced the cell death induced by fixed anti-CD3 monoclonal antibody (mAb), especially when a high concentration of anti-CD3 mAb was used. DNA fragmentation increased in parallel with cell death, indicating that cell death was a result of the apoptosis. Fluorescence-activated cell sorter (FACS) analysis revealed that the activation-induced cell death (AICD) caused by anti-CD3 mAb alone, or by a combination of anti-CD3 mAb and FN, occurred selectively in CD4+ CD8+ thymocytes. Very late activation antigen (VLA)-4 and VLA-5 are two major ligands to FN on thymocytes. The expression of both ligands was investigated at different stages of thymocyte development. VLA-4 was predominantly expressed at the CD4- CD8- stage, and thereafter the expression was reduced, whereas VLA-5 was constantly expressed during maturation. Furthermore, the enhancing effect by FN was inhibited in the presence of the Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide but not in the presence of the connecting segment-1 (CS-1) peptide, suggesting that enhancement of AICD observed in CD4+ CD8+ thymocytes is mediated through VLA-5.