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The protein expression system in bacteria is widely used to overproduce recombinant proteins. Many established expression vectors are on the market. The Escherichia coli expression system is extremely useful for biochemical and biophysical analysis. Here, we describe the results of the expression of mammalian protein in E. coli and an experiment on the structural analysis of recombinant protein.

Cysteine (cysteinyl residue) modifications in proteins result in diversity in protein functions. The reaction specificity of a protein with a modified cysteine residue is determined by the overall conditions of the protein, including the spatial position of the cysteine residue, electrostatic interactions between cysteine residue and other charged residues, spatial interactions between the cysteine residue and a chemical compound, electrophilicity of the chemical compound, and the pH of the solution. In cysteine-dependant enzymes, each specific type of cysteine modification characterizes the catalytic mechanism of the enzyme. Recently, the catalytic mechanisms of peroxiredoxins and cysteine proteases, which contain a cysteine residue(s) in their catalytic sites, have been elucidated. In the catalytic process of peroxiredoxins, a sulfenyl intermediate is formed by oxidation of the catalytic cysteine residue. On the other hand, in cysteine proteases, the catalytic cysteine residue reacts with the carboxyl carbon of a peptide substrate to form an intermediate complex via S-alkylation. In this review, we introduce the most current information on the applications of cysteine thiol chemistry for in vitro glycoprotein synthesis. Recently, a glycoprotein (monocyte chemotactic protein-3), containing an intact human complex-type sialyloligosaccharide has been chemically synthesized. The procedure used for this could have applications in the development of new protein-based drugs, including antineoplastic drugs and antibiotics. It can also potentially be applied for improving the half-life and reducing the toxicity of these drugs, and for preventing the development of multidrug resistance.

Protein cysteines (cysteinyl residues) play critical roles in biological processes. In the course of protein evolution under oxidizing atmosphere of the Earth, organisms have utilized highly reactive cysteines in many proteins essential for maintenance of life, i.e. enzymes, transcriptional factors, cytoskeletons, and receptors. In some enzymes, sophistical cysteine modification characterizes each catalytic mechanism. In vivo modification of protein cysteines with natural chemical compounds modulates protein functions as a molecular switch. Oxidation/reduction, thiol-disulfide exchange, nitrosylation, sulfuration, thiolation, acylation and prenylation are involved. Some protein cysteines coordinate metals or metal cofactors such as a heme or an iron sulfur cluster to form metalloproteins, serving as sensor proteins, metalloenzymes or transcriptional factors. Information on the in vitro chemical modifications and their reaction specificities of protein cysteines are essential for the investigation of the mechanisms and functions of in vivo protein cysteine modifications. In this review, we also mention historically important knowledge other than recent results on protein cysteine modification and modulation of protein function to fertilize medical proteomics.

Proteomic analysis of inner ear proteins revealed unique properties of cochlin, encoded by the COCH gene. We detected 3 cochlin isoforms, p63s, p44s and p40s, in the inner ear tissue and a short 16-kDa isoform, cochlin-tomoprotein (CTP), in the perilymph. The role of the cochlin isoforms has not been elucidated. To improve our understanding of the mechanism of cochlin isoform expression, we investigated rat cochlin mRNA expression in the inner ear and other organs. We performed RNA-ligation-mediated amplification of cDNA ends (RLM-RACE) using RNA isolated from the inner ear and spleen of rats, which are known to express abundant cochlin mRNA. We also examined the expression profile of full-length cochlin mRNA by nested RT-PCR in the cerebrum, cerebellum/brain stem, eye, inner ear, thyroid gland, thymus gland, lung, heart, liver, spleen, adrenal gland, kidney and blood. We verified CTP expression in rat perilymph by Western blot. By RLM-RACE, alternately spliced variants of cochlin mRNA with 3 different lengths were detected (2442, 2008 and 724 bp). The two longer mRNAs encode full-length cochlin with different polyadenylation signals in the 3′-untranslated region, which are expressed both in the ear and spleen. The short variant encodes the limulus factor C, cochlin, late gestation lung protein (LCCL) domain and the N-terminal sequence of the von Willebrand factor A (vWFA1) domain, and this variant was detected only in the ear. All 3 variants have the same transcriptional start site. By RT-PCR, we found that full-length cochlin was expressed in all organs examined, with a splice variant in the heart. By Western blot, we detected short isoforms (11–17 kDa) in the perilymph. Cochlin isoform formation is regulated, at least in part, by alternative splicing at the transcriptional level. The short mRNA was detected only in the inner ear, and this variant may provide a clue to understanding the formation and function of cochlin isoforms.

Resveratrol ( trans-3,4',5-trihydroxystilbene) is a natural phytoalexin with various biological activities including inhibition of lipid peroxidation and free radical scavenging properties. In addition to its beneficial effects, resveratrol also has significant genotoxicity that leads to a high frequency of chromosome aberration together with micronucleus and sister chromatid exchanges. To enhance the radical scavenging activities and to reduce the genotoxicity of resveratrol, we designed 4'-methyl resveratrol analogues where a methyl group was introduced at the ortho position relative to the 4'-hydroxy group, which is responsible for both antioxidative activities and genotoxicity of resveratrol. These synthesized methyl analogues of resveratrol showed increased antioxidative activities against galvinoxyl radical as an oxyl radical species. Furthermore, the methyl analogues also surprisingly showed reduced in vitro genotoxicities, suggesting that methyl substitution may improve resveratrol efficacy.

Rat heme-binding protein 23 (HBP23)/peroxiredoxin (Prx I) belongs to the 2-Cys peroxiredoxin type I family and exhibits peroxidase activity coupled with reduced thioredoxin (Trx) as an electron donor. We analyzed the dimer-oligomer interconversion of wild-type and mutant HBP23/Prx I by gel filtration and found that the C52S and C173S mutants existed mostly as decamers, whereas the wild type was a mixture of various forms, favoring the decamer at higher protein concentration and lower ionic salt concentration and in the presence of dithiothreitol. The C83S mutant was predominantly dimeric, in agreement with a previous crystallographic analysis (Hirotsu, S., Abe, Y., Okada, K., Nagahara, N., Hori, H., Nishino, T., and Hakoshima, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12333-12338). X-ray diffraction analysis of the decameric C52S mutant revealed a toroidal structure (diameter, approximately 130A; inside diameter, approximately 55A; thickness, approximately 45A). In contrast to human Prx I, which was recently reported to exist predominantly as the decamer with Cys(83)-Cys(83) disulfide bonds at all dimer-dimer interfaces, rat HBP23/Prx I has a Cys(83)-Cys(83) disulfide bond at only one dimer-dimer interface (S-S separation of approximately 2.1A), whereas the interactions at the other interfaces (mean S-S separation of 3.6A) appear to involve hydrophobic and van der Waals forces. This finding is consistent with gel filtration analyses showing that the protein readily interconverts between dimer and oligomeric forms. The C83S mutant exhibited similar peroxidase activity to the wild type, which is exclusively dimeric, in the Trx/Trx reductase system. At higher concentrations, where the protein was mostly decameric, less efficient attack of reduced Trx was observed in a [(14)C]iodoacetamide incorporation experiment. We suggest that the dimerdecamer interconversion may have a regulatory role.

Although placental proteins play multiple roles in fetal and placental development and in the maintenance of pregnancy, many remain inadequately characterized. In the present study, we comprehensively analyzed these proteins by using a proteomic approach. Samples were denatured with guanidine hydrochloride, which was found to be superior to the commonly used urea for the present purpose, and subjected to 2-dimensional (2D) electrophoresis (2-DE) to obtain placental proteome maps. The identified protein spots (ca. 60% of the total) on the proteome maps included several pregnancy-related proteins (PRPs). Furthermore, a novel 2D immunoblotting (2-DI) analysis of molecules related to pre-eclampsia revealed three immunopositive spots that appeared to correspond to dynactin p-50, a protein related to cell turn-over. The rate of positivity for dynactin p-50-reactive antibodies was significantly (P = 0.0024) higher in 26 pre-eclamptic women than in 58 normally pregnant women. These results indicate that dynactin p-50 may be involved in the pathophysiology of pre-eclampsia.

Rat 3-mercaptopyruvate sulfurtransferase (MST) contains three exposed cysteines as follows: a catalytic site cysteine, Cys247, in the active site and Cys154 and Cys263 on the surface of MST. The corresponding cysteine to Cys263 is conserved in mammalian MSTs, and Cys154 is a unique cysteine. MST has monomer-dimer equilibrium with the assistance of oxidants and reductants. The monomer to dimer ratio is maintained at ∼92:8 in 0.2 m potassium phosphate buffer containing no reductants under air-saturated conditions; the dimer might be symmetrical via an intersubunit disulfide bond between Cys154 and Cys154 and between Cys263 and Cys263, or asymmetrical via an intersubunit disulfide bond between Cys154 and Cys263. Escherichia coli reduced thioredoxin (Trx) cleaved the intersubunit disulfide bond to activate MST to 2.3- and 4.9-fold the levels of activation of dithiothreitol (DTT)-treated and DTT-untreated MST, respectively. Rat Trx also activated MST. On the other hand, reduced glutathione did not affect MST activity. E. coli C35S Trx, in which Cys35 was replaced with Ser, formed some adducts with MST and activated MST after treatment with DTT. Thus, Cys32 of E. coli Trx reacted with the redox-active cysteines, Cys154 and Cys263, by forming an intersubunit disulfide bond and a sulfenyl Cys247. A consecutively formed disulfide bond between Trx and MST must be cleaved for the activation. E. coli C32S Trx, however, did not activate MST. Reduced Trx turns on a redox switch for the enzymatic activation of MST, which contributes to the maintenance of cellular redox homeostasis.

Three-dimensional unsteady flow with localized areas with large vortices in the intake channel of a small-scale hydroelectric power station has been simulated numerically using a Large-Eddy Simulation (LES) technique. The method computes the motion of the free surface and a standard Smagorinsky model with wall damping is used as the turbulence model. With the total number of grid points of about one-third of a million, the complex and unsteady nature of the vortical flow can be reproduced reasonably well by marginally resolving the near-wall flow. Intake vortex of weak intensity can be captured and the flow conditions associated with appearance of vortices can be examined based on the results of the present simulation.

The purpose of this study was to investigate the association of the expression of vascular endothelial growth factor-C (VEGF-C) with regional lymph node metastasis in oral squamous cell carcinoma (OSCC). The expression of VEGF-C in biopsy specimens obtained from 62 patients with OSCC was examined by immunohistochemistry. In the early stages of T1 and T2 (38 cases), VEGF-C expression strongly correlated with lymph node metastasis (P

口腔外科領域の末期癌患者は原発巣や頸部転移巣からの出血により死亡することがあり, そのために患者のQOLの著しい低下を招くことがある。今回我々は口腔癌の末期で原発巣や頸部転移巣から出血した患者に対して動脈塞栓術を施行した3例を経験したのでその概要を報告する。さらに塞栓術4日後に死亡した1例を除く2例について塞栓術前後のKarnofsky Performance Statusscale (KPS) と血清アルブミン値について比較検討した。その結果KPS, 血清アルブミン値は改善しQOLも改善したと考えられた。今回の経験から腫瘍から出血を生じた口腔癌の末期患者に対する塞栓術が患者のQOLの維持, 改善と延命につながると考えられた。

In this study, we examined the expression of cyclins, cyclin dependent kinase (CDKs) and CDK inhibitors by immunohistochemical analysis in 20 normal mucosa, 42 epithelial dysplasia (ED), and 117 oral squamous cell carcinoma. Neither Cyclin D1 nor CDK2 were detectable in normal tissue and ED. Their presence, however, was detectable in squamous cell carcinoma (SCCs) (Cyclin D1, 35.9%; CDK2, 66.7%). Cyclin E was detectable in 57.1% of severe ED and 62.8% of SCCs. For the CDK inhibitors, these proteins were detectable in all normal mucosa and most of the mild and moderate ED. For severe ED, expression of these proteins was not observed in some cases (p12(DOC-1), 14.3%; p16(INK4A), 28.6%; p27(KIP1), 7.1%). For SCCs, the expression of p12(DOC-1) was lost in 71.8%, p16(INK4A) in 69.2% and p27(KIP1) in 35.9%. These results suggest that elevated expression of cyclin D1, cyclin E, CDK2 and loss of p12(DOC-1), p16(INK4A) and p27(KIP1) may contribute to the multistep nature of oral carcinogenesis.

Basal cell nevus syndrome is a complex disease characterized by multiple cysts of the jaws, multiple basal cell tumors of the skin, and a high incidence of skeletal abnormalities. Jaw cysts often involve permanent teeth in young patients, and we have to preserve permanent teeth whenever possible. We encountered a 9-year-old boy who had multiple jaw cysts and treated him for 9 years. It was possible to preserve the permanent teeth affected by jaw cysts by means of marsupialization. No growth disturbance of the jaws occurred.

Epidermal growth factor receptor (EGFR) regulates the growth and progression of human oral squamous cell carcinoma (SCC). Recently, the link between EGFR signaling and the cell cycle has been identified. Some reports have described that EGFR-blocking monoclonal antibody 225 (mAb225) induced G1 arrest and inhibited the growth of various cancer cells. The purpose of this study was to evaluate the effect of mAb225 on human oral SCC cell lines. Exposure to mAb225 in culture inhibited the growth of oral SCC cell lines in an EGFR number-independent manner, with the percent inhibition ranging from 13.8 to 76.6%. Flow-cytometric analysis demonstrated that treatment with mAb225 induced cell accumulation in G1 phase, accompanied by a decrease in the percentage of cells in the S phase. Apoptosis was not seen in this study. G1 arrest was accompanied by a decrease in CDK2-, CDK4-, and CDK6-associated histone H1 kinase activities, and an increase in the expression levels of cell cycle inhibitors p27 KIP1 and p15 INK4B . These results suggested that the antiproliferative effect of EGFR blockade by mAb225 in oral SCC may be mediated by p27 KIP1 and p15 INK4B .

岡山大学歯学部附属病院第二口腔外科にて加療した口腔悪性黒色腫4例について臨床的検討を行った。対象症例の性別は男性2例, 女性2例, 年齢は57歳から75歳までであった。原発部位は下顎歯肉1例, 口蓋3例で初診時のUICC (1997) 分類はstage Ia期: 1例, stage II期: 1例, stage III: 2例であった。治療法は全例外科療法を施行し, 化学療法及び免疫療法またはホルモン療法を併用した。局所再発及び頸部リンパ節転移または遠隔転移をきたした2例が死亡例であった。他の2例は, 局所再発及び転移はなく, 経過観察期間は14年11ケ月と3年8ケ月であった。

1982年4月より1999年3月までに岡山大学歯学部附属病院第二口腔外科を受診し, 病理組織学的に粘表皮癌と診断された27例について臨床統計的検討を行ったので報告する。 (1) 性別発生頻度は, 男性16例, 女性11例。 (2) 年齢別発生頻度は平均63.0歳であった。 (3) 部位別発生頻度をみると大唾液腺2例, 小唾液腺25例。 (4) TNM分類ではT1が7例, T2が7例, T4が13例であり, Stage別では, Stage Iが7例, Stage IIが7例, Stage IVが13例であった。 (5) 治療法については, 24例に対して外科療法を施行し, そのうち外科療法の単独施行は13例, 化学療法および放射線療法の併用療法を施行したものは11例であった。 (6) 全体の5年累積生存率は69.4%で, Stage I, II症例が90.9%, Stage IV症例が46.4%であった。

過去12年間の頭頸部再建術は約365例であり, 島状皮弁48例に対し, マイクロサージャリーによる再建術は300例であった。主な再建材料の内訳は, 有茎移植組織は, 顔面神経麻痺の再建としての側頭筋移行, 再発癌に対する拡大型広背筋皮弁が主であった。遊離組織移植は, 前大腿皮弁または大腿筋膜張筋皮弁 (TFL flap) (89 flaps), DIEP flap などの腹壁皮弁 (48 flaps), 橈側前腕皮弁 (28 flaps), シャム型大腿筋膜張筋―前外側大腿連合皮弁 (18 flaps) などであった。また, 皮弁採取部の筋, 主要動脈を最大限温存すべく腹直筋皮弁, 広背筋皮弁, 橈側前腕皮弁から腹壁穿通枝皮弁, 広背筋穿通枝皮弁, 橈骨動脈穿通枝皮弁, 上腕皮弁などの穿通枝皮弁に移行しつつある。時に特殊な例では大伏在静脈皮弁を用いる。さらに最近は0.7mm前後の血管吻合 (supermicrosurgery) が可能となり頭頸部に有用な新たな組織 (部分耳介による眼瞼や気管全周性欠損の再建, 趾間皮弁による口角部再建, オトガイ下皮弁による口唇部再建, 腹部脂肪弁による顔面陥凹変形再建など) 移植による再建がなされつつある。

Connective tissue growth factor (CTGF) is known to be a potent angiogenic factor. Here, we present the evidence that the hypoxic induction of angiogenesis by human breast cancer cells (MDA-231) can be ascribed at least in part to CTGF. Our results indicate that (i) CTGF is abundantly present in MDA-231 cells in vitro and in vivo, (ii) its secretion is up-regulated by hypoxia, and (iii) its gene expression is enhanced in MDA-231 cells cultured under hypoxic conditions. These data suggest CTGF may stimulate angiogenesis by paracrine mechanisms, thereby contributing to the invasion of breast cancer cells. This is the first evidence that human cancer cells differentially express CTGF protein and mRNA under the control of hypoxic conditions.

Objective. The purpose of this study was to discriminate radiographically between dentigerous cysts (DCs) and odontogenic keratocysts (OKCs) associated with a mandibular third molar. Study design. The material consisted of panoramic radiographs of dentigerous cysts (44 patients, 45 cysts) and odontogenic keratocysts (15 patients, 16 cysts), all of which were related to a mandibular third molar. The radiographic images were analyzed with reference to the patients’ ages and symptoms. Results. The mean age of patients in the OKC group was less than that of patients in the DC group. The mean area of the cysts in the OKC group was larger than that of those in the DC group. The mean distance from the second to the third molar in the DC group was greater than that in the OKC group. Although there was a significant correlation between the area and distance in the DC and OKC groups, the patients’ ages did not significantly correlate to the area and distance of either cyst. Conclusions. The OKCs had a tendency toward rapid growth in the patient’s youth but short movement of a third molar compared with the DCs. The DCs and OKCs do not appear to develop gradually from the period when follicles or dental lamina were formed but arise at various periods randomly. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:743-7)

Many studies focused on the tumour thickness in oral squamous cell carcinomas, suggesting a relationship with the occurrence of cervical metastasis. Accurate preoperative assessment of the tumour thickness of oral cancer would provide useful information for targeting those patients who need elective treatment of the neck. Some useful diagnostic aids to evaluate oral cancer are computed tomography (CT), magnetic resonance imaging (MRI), and intraoral ultrasonography. The purpose of the present study is to compare intraoral ultrasonography with CT and MRI in delineating the disease extent and in measuring the tumour thickness of oral carcinoma. Thirty-nine patients with oral cancer were preoperatively evaluated with intraoral ultrasonography, and CT, and in 26 of them MRI was carried out. High-quality ultrasonographic images were obtained and the tumour thickness was measured within 1 mm. However, in most tumours less than 5.0 mm in thickness, CT and MRI could not detect a sufficient density difference from the normal tissue to accurately delineate the extent of the tumour. There was a significant correlation between measurements by intraoral ultrasonography and the histological sections. The present study shows that ultrasonography is superior to CT and MRI in assessment of the primary lesion of oral carcinoma.

Cyclins and cyclin-dependent kinases (CDKs) play key roles in cell cycle regulation, a process of which dysregulation can lead to uncontrolled cell growth and hence to cancer. We have already reported the alteration of CDK4 and cyclin D1 expression in oral cancer. In this study, we examined by immunohistochemistry the expression of CDK2, and cyclins A and E in 20 normal oral mucosa, 42 dysplastic epithelia, and 103 oral squamous cell carcinomas (SCCs). The expressions of CDK2, and cyclins A and E were not detected in the normal epithelium and significantly altered from epithelial dysplasia to SCC. While there were no significant correlations between the expression of cyclins A, E and the patients' survival, CDK2 expression was significantly correlated with lymph node involvement (P = 0.025), tumor differentiation (P = 0.032), mode of tumor invasion (P = 0.017), and shorter survival period (P = 0.0173). These results suggest that the elevated expression of CDK2 is a critical factor in oral cancer progression and can be used as a negative predictive marker of the patients' prognosis.

p16 INK4A inactivation was analyzed in ten squamous cell carcinoma (SCC) cell lines and 32 primary SCCs, using the polymerase chain reaction (PCR), PCR–single-strand conformation polymorphism, methylation-specific PCR, and cycle sequencing. In the study of cell lines, we detected three deletions in exon 1α and exon 2, and detected two methylations. Among tumor samples, we detected the homozygous deletions (HDs) of 43.8% in exon 1α 34.4% in exon 2, and methylation was found in 50.0%. The lack of p16 INK4A with immunohistochemistry was detected in 71.9% and matched the alteration of p16 INK4A gene. These results suggest that p16 INK4A inactivation is predominantly caused by HD and methylation, and immunohistochemical evaluation of p16 INK4A is a useful method.

Initial tissue response to anti-washout apatite cement (aw-AC) in the palatal region was studied. Conventional apatite cement (c-AC) was employed as a control material. Bone defects generated in the rat palatal region, where complete hemostasis is difficult to effect, were filled with both cement types and examined histologically for up to 8 weeks. At 1-week postfilling, a portion of the c-AC had washed out, resulting in slight inflammation and severe foreign-body response. The degree of foreign-body response to c-AC was reduced over time; however, foreign-body response continued to be in evidence 8 weeks after surgery. As a result, poor bone formation was observed in the case of c-AC at 8 weeks post-surgery. In contrast, aw-AC set well, maintained its shape at implantation, and caused little foreign-body response. Osteoblasts were observed at 2 weeks following surgery. Moreover, the bone defect was completely covered with new bone at 8 weeks post-surgery. This observation suggests that aw-AC may be used without complication in cases where complete hemostasis is difficult to achieve, that is, where the use of c-AC is contraindicated.

This study examined the relationship between tumor angiogenesis and the radiation-induced response, evaluated based on pathological changes, in oral squamous cell carcinoma patients treated with preoperative radiation therapy. Forty-one cases of squamous cell carcinoma treated with preoperative radiation therapy were investigated. Tumor angiogenesis was assessed by scoring the intratumor microvessel density (IMVD). Expression of vascular endothelial growth factor (VEGF) was also evaluated before and after preoperative radiotherapy. There was no correlation between IMVD in the specimens before therapy and the pathological response to radiation therapy. However, radiation therapy decreased IMVD in the specimens after therapy. A significant association was observed between VEGF expression and resistance to radiation therapy: only 4 of the 21 patients whose tumors exhibited a high level (2 + or 3 + ) of VEGF staining experienced a major (3 + or 4 + ) pathological response to radiation therapy. Furthermore, an increasing level of VEGF expression after radiation therapy was observed in non-effective (0 to 2 + ) response cases. These results suggest that VEGF expression and the induction of this protein are related to radiosensitivity and could be used to predict the effects of preoperative radiation therapy on oral squamous cell carcinoma.

Purpose: Cisplatin (cis-diamminedichloroplatinum(II), CDDP) has been reported to induce apoptosis in cancer cells, the mechanism of the apoptosis in cancer cells induced by CDDP is still unclear. Recent studies have revealed that caspase family of cystine proteases play an important role in the regulation of several apoptotic processes. In this study, whether apoptosis induced by CDDP could be mediated by the activation of caspase-3, a caspase family protease, was investigated. Methods: The CDDP-resistant subline A431/CDDP2 from the previously established human epidermoid carcinoma cell line A431 was used. The parent A431 cells (A431/P) and the A431/CDDP2 were exposed to CDDP with or without a caspase family protease inhibitor (Z-Asp-CH2-DCB), and cellular sensitivity to CDDP was determined. DNA fragmentation was then analyzed, and the caspase-3 protein levels determined by Western blotting following exposure of the cells to CDDP with or without Z-Asp-CH2-DCB. Results: In the A431/P cells, the cytotoxicity of CDDP was clearly reduced by Z-Asp-CH2-DCB compared with its cytotoxicity in A431/CDDP2 cells. Furthermore, quantitative analysis of DNA fragmentation revealed that Z-Asp-CH2-DCB inhibited DNA fragmentation induced by CDDP in A431/P cells, but not in A431/CDDP2 cells. Western blotting analysis demonstrated a marked reduction in procaspase-3 protein levels in A431/P cells treated with Z-Asp-CH2-DCB. In the A431/CDDP2 cells, procaspase-3 protein levels were no different with and without Z-Asp-CH2-DCB. Conclusions: These findings suggest that caspase-3 may mediate apoptosis induced by CDDP, and its induction could represent a novel approach to the effective treatment of malignant tumors.

This study was carried out to elucidate whether apoptosis and p53 can be used to stratify oral cancer patients into groups with a favorable or unfavorable response to preoperative radiation therapy. Thirty-two patients were evaluated. The apoptosis index was 1.7+/-0. 9% in the ineffective cases, and it was significantly lower than effective cases (3.2+/-1.2%). While 14 of 16 effective cases (86.7%) did not express p53, 13 of 16 ineffective cases (81.3%) overexpressed p53. These results suggest that mutated p53 in tumors is associated with a poor response to radiation which may be related to evasion of apoptosis in oral cancer.

Background. The intraarterial approach is one of the most important routes for the administration of anticancer drugs for head and neck cancer. A profound knowledge of the anatomical characteristics and variations of the carotid artery, such as its branching pattern, length, and inner diameter, is essential to avoid complications with catheter insertion. Methods. We conducted a morphometric investigation of head and neck arteries in 29 Japanese cadavers (58 sites). Results. The branching pattern of the external carotid artery showed variations. In 65.5% of the cadavers, the lingual, facial, and superior thyroid arteries arose separately. However, in 31.0% of the cadavers, the lingual artery formed a common trunk with the facial artery, and in 3.5%, the lingual artery formed a common trunk with the superior thyroid artery. The transverse facial artery arose from the superficial temporal artery in 53.4% of the specimens, from the maxillary artery in 27.6%, and from a site central to the maxillary artery in 19.0%. The posterior auricular artery arose from the external carotid artery at the same level as the maxillary artery in 37.9% of specimens, and from a site central to the maxillary artery in 62.1%. The occipital artery arose from the external carotid artery at the same level as the maxillary artery in 55.2% of specimens, and from a site peripheral to the facial artery in 44.8%. The lengths from the auricular point to the origins of the upper branches of the external carotid artery were: 2.8 mm to the transverse facial artery, 3.2 cm to the maxillary artery, 3.8 cm to the posterior auricular artery, 6.6 cm to the occipital artery, 7.4 cm to the facial artery, 8.8 cm to the lingual artery, and 10.4 cm to the superior thyroid artery. Conclusions. These results, have led to some clarification of the clinicoanatomical basis for intraarterial infusion. These data should be helpful for assessing the approximate level of the catheter tip and for evaluating whether the catheter is placed appropriately, by transient staining of the infused area.

The mechanism of osteolysis associated with metastatic cancer of the jaws is essentially osteoclast-mediated. Therefore, it is likely that potent osteoclastic bone resorption inhibitors such as bisphosphonates would be efficacious for the treatment of jaw metastasis. We examined the effects of a third generation bisphosphonate, YM175, in a nude mice jaw metastasis model with intracardiac injection of a human breast cancer cell line, MDA-MB-231. The metastatic lesions in untreated mice were radiographically observed at the body and angle of the mandible. Histology of the mandible of untreated mice revealed that most of the bone marrow cavities had been occupied by the metastatic tumor with active osteoclasts along the trabecular bone. The experimental group showed that YM175 markedly reduced the size of tumor and the number of osteoclasts. These results suggest that YM175 may suppress metastasis formation and tumor growth in jaw through inhibition of osteoclastic bone resorption.

Connective tissue growth factor (CTGF) is a novel cysteine-rich, secreted protein. Recently, we found that inhibition of the endogenous expression of CTGF by its antisense oligonucleotide and antisense RNA suppresses the proliferation and migration of vascular endothelial cells. In the present study, the following observations demonstrated the angiogenic function of CTGF in vitro and in vivo: (i) purified recombinant CTGF (rCTGF) promoted the adhesion, proliferation and migration of vascular endothelial cells in a dose-dependent manner under serum-free conditions, and these effects were inhibited by anti-CTGF antibodies; (ii) rCTGF markedly induced the tube formation of vascular endothelial cells, and this effect was stronger than that of basic fibroblast growth factor or vascular endothelial growth factor; (iii) application of rCTGF to the chicken chorioallantoic membrane resulted in a gross angiogenic response, and this effect was also inhibited by anti-CTGF antibodies. (iv) rCTGF injected with collagen gel into the backs of mice induced strong angiogenesis in vivo. These findings indicate that CTGF is a novel, potent angiogenesis factor which functions in multi-stages in this process.

Glutamate synthase (GOGAT) is a key enzyme in the assimilation of inorganic nitrogen in photosynthetic organisms. We found that, like higher plants, the facultative heterotrophic cyanobacterium Plectonema boryanum had ferredoxin (Fd)- and NADH-dependent GOGATs. The genes glsF, gltB, and gltDwere cloned, and structural analyses and target mutageneses demonstrated that glsF encoded Fd-GOGAT and thatgltB and gltD encoded the two subunits of NADH-GOGAT. All three mutants lacking one of the GOGAT genes were able to grow photosynthetically and heterotrophically. However, the Fd-GOGAT mutant exhibited a phenotype of marked nitrogen deficiency when grown under conditions of saturating illumination and CO2 supply. In these conditions the rate of the ammonia uptake from the culture medium was slower in the Fd-GOGAT mutant than in the wild type or in the NADH-GOGAT mutant, but no significant differences were found in the rate of the CO2 fixation-dependent O2 evolution among these strains. Our results suggest that, although both Fd- and NADH-GOGATs were operative in the cells growing in light, the contribution of Fd-GOGAT, which directly utilizes photoreducing power for the catalytic reaction, is essential for balancing photosynthetic nitrogen and carbon assimilation.

In maize (Zea mays L.) two leaf-specific ferredoxin (Fd) isoproteins, Fd I and Fd II, are distributed differentially in mesophyll and bundle-sheath cells. A novel cDNA encoding the precursor of Fd II (pFD2) was isolated by heterologous hybridization using a cDNA for Fd I (pFD1) as a probe. The assignment of the cDNAs to the Fds was verified by capillary liquid-chromatography/electrospray ionization-mass spectrometry. RNA-blot analysis demonstrated that transcripts for Fd I and Fd II accumulated specifically in mesophyll and bundle-sheath cells, respectively. The mature regions of pFD1 and pFD2 were expressed in Escherichia coli as functional Fds. Fd I and Fd II had similar redox potentials of −423 and −406 mV, respectively, but the Km value of Fd-NADP+ reductase for Fd II was about 3-fold larger than that for Fd I. Asparagine at position 65 of Fd II is a unique residue compared with Fd I and other Fds from various plants, which have aspartic acid or glutamic acid at the corresponding position as an electrostatic interaction site with Fd-NADP+ reductase. Substitution of asparagine-65 with aspartic acid increased the affinity of Fd II with Fd-NADP+ reductase to a level comparable to that of Fd I. These structural and functional differences of Fd I and Fd II may be related to their cell-specific expression in the leaves of a C4 plant.

YH529, [1-hydroxy-2-(imidazo [1,2-a] pyridin-3-yl) ethylidene]-bisphosphonic acid monohydrate, is a newly developed third-generation bisphosphonate with a potent inhibitory activity toward osteoclastic bone resorption. The primary cellular mechanism of osteolysis associated with metastatic cancer is osteoclast-mediated. It is likely that bisphosphonates would be efficacious in this situation. In the present study, we examined the effect of YH529 in a nude mice bone metastasis model, in which the intracardiac injection of a human breast cancer cell line, MDA-MB-231(MDA-231), leads to osteolytic bone metastases. To examine whether YH529 would prevent such bone metastasis, we administered YH529 s.c. to nude mice simultaneously with cancer cell inoculation through the entire experimental period (protocol 1) or performed short-term prophylactic administration before inoculation of the MDA-231 cells (protocol 2). In addition, to examine the possible therapeutic effects of the drug on established bone metastases, we injected YH529 after radiographically small but distinct osteolytic bone metastases had been detected (protocol 3). In all protocols, YH529 (2 μg/mouse/day) markedly inhibited bone metastases as well as the progression of established metastatic foci that were quantified on the radiographs. Histological examination and histomorphometrical analysis revealed that YH529 markedly reduced the number of osteoclasts and the size of the tumor at the metastatic bone sites. Our results suggest that YH529 may suppress metastasis formation and tumor growth in bone through inhibition of osteoclastic bone resorption. Int. J. Cancer 77:279–285, 1998.© 1998 Wiley-Liss, Inc.

Previously, we cloned an mRNA predominantly expressed in hypertrophic chondrocytes by differential display-PCR from a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) that is identical to that of connective tissue growth factor (CTGF). In the present study, we investigated the roles of CTGF in the proliferation and migration of vascular endothelial cells using its antisense oligonucleotide and antisense RNA, because angiogenesis into the hypertrophic zone of cartilage occurs at the final step of endochondral ossification. Immunohistochemical and immunofluorescence techniques revealed that not only hypertrophic chondrocytes but also endothelial cells in the cost-chondral junctions of mouse ribs were stained with an anti-CTGF antibody in vivo. Northern blot analysis revealed that CTGF was strongly expressed in chondrocytic cells as well as bovine aorta endothelial (BAE) cells in culture, but not in other types of cells such as osteoblastic cells. Its expression in BAE cells was greater in the growing phase than in the confluent phase. When one-half of a monolayer of a confluent culture of BAE cells had been peeled off, only the cells proliferating and extending into the vacant area were stained with the anti-CTGF antibody. The addition of an antisense oligonucleotide inhibited the proliferation and extension of the BAE cells into the vacant area. The antisense oligonucleotide also inhibited the proliferation of BAE cells in the rapidly proliferating phase. In a Boyden chamber assay, pretreatment with the antisense oligonucleotide markedly inhibited the migration of BAE cells. Furthermore, the abilities to proliferate and migrate of BAE cells, which were stably transfected with expression vectors that generate the antisense RNA of CTGF cDNA, were markedly lower than those of the control. These findings suggest that endogenous CTGF expression is involved in the proliferation and migration of BAE cells.

doc-1 is a putative tumor suppressor gene isolated and identified from the hamster oral cancer model. Here, we report the molecular cloning and the functional characterization of the human ortholog of the hamster doc-1 gene. Human doc-1 cDNA is 1.6 kilobase pairs in length and encodes for a 115-amino acid polypeptide (12.4 kDa, pI 9. 53). Sequence analysis showed 98% identity between human and hamster doc-1 protein sequences. DOC-1 is expressed in all normal human tissues examined. In oral keratinocytes, expression of DOC-1 is restricted to normal oral keratinocytes. By immunostaining of normal human mucosa, DOC-1 is detected in both the cytoplasm and nuclei of basal oral keratinocytes; while in suprabasilar cells, it is primarily found in the nuclei. Human oral cancers in vivo did not exhibit immunostaining for DOC-1. Like murine DOC-1, human DOC-1 associates with DNA polymerase alpha/primase and mediates the phosphorylation of the large p180 catalytic subunit, suggesting it may be a potential regulator of DNA replication in the S phase of the cell cycle. Using a human doc-1 cosmid as a probe, human doc-1 is mapped to chromosome 12q24. We identified four exons in the entire human doc-1 gene and determined the intron-exon boundaries. By polymerase chain reaction and direct sequencing, we examined premalignant oral lesion and oral cancer cell lines and found no intragenic mutations.

Amplification of the c-erbB-3 gene and the expression of its mRNA and protein in human squamous cell carcinomas (SCC) were analyzed. Although no amplification of the c-erbB-3 gene was observed, overexpression of its mRNA was observed in SCC by RT-PCR. The expression of this gene in SCC was 15–50 times higher than in normal fibroblasts. Overexpression of the secreted type of erbB-3 mRNA was also observed in all SCC. Moreover, overproduction of the c-erbB-3 protein was also detected in SCC by dot blot analysis using anti-c-erbB-3 monoclonal antibodies. In nude mice, the expression of c-erbB-3 mRNA was higher in metastatic tumors compared with primary tumors. These results suggest that both the transmembrane and secreted types of c-erbB-3 play a significant role in the formation and development of SCC.

Malignant tumors are composed of cells with different phenotypic properties and only certain cell subpopulations present metastatic potential. The establishment of cell lines with high or low metastatic potential is necessary to investigate the molecular mechanisms of the metastatic process. However, human oral squamous cell carcinoma (SCC) cell lines that are suitable for the above investigation are scarce. High and low metastatic cells were obtained from a primary lesion of a patient with tongue carcinoma who had not received any therapy. Two distinct cell lines were selected, UM1 with a scattered growth pattern and loose cell-cell adhesion, and UM2 with a colony-formed growth pattern and firm cell-cell adhesion. The expression of E-cadherin in UM1 was clearly lower than that in UM2. UM1 exhibited a higher motility, invasive and metastatic activity than UM2 in vivo and in vitro. A low invasive and a metastatic oral SCC cell line, useful to investigate invasion and metastasis mechanisms, have been established.

本論文は, 口腔癌患者におけるVideofluoro-graphyを用いた嚥下の評価に関する経験について述べた。17例の口腔癌患者を対象に検査を行った。嚥下の様相は, 術前ならびに術後の摂食開始時にVF検査にて評価され, 記録された。VFによる評価により臨床的な観察によって同定されない異常所見を見いだすことができた。患者とともにVF検査結果を基に嚥下訓練について話し合うことで, 患者の協力が得られた。以上のことから, VFによる評価は嚥下様相の理解とともに嚥下機能訓練に有用な方法と考えられた。

To evaluate mechanisms of the growth of Asteraceae plants by osmoregulation under saline conditions, ions uptake, accumulation of compatible solutes, and osmotic potential were measured in leaves of glycophytes safflower (Carthamus tinctorius L.'Roundish Leaf', salt sensitive), Chrysanthemum paludosum Poir. 'North Pole'(moderate tolerance), and a halophyte sea aster (Aster tripolium L., high tolerance) grown under hydroponics including NaCl. Salt stress was imposed for 16 days by adding 12.5, 25, or 50 mM NaCl into nutrient solutions for safflower and C. paludosum, and 75, 150, or 300 mM NaCl for sea aster. Leaf osmotic potentials decreased and the uptakes of Na+ and Cl- increased with increases in NaCl for all species. In C. paludosum and sea aster, Na+ concentration in the shoot was significantly higher than that in the root, showing that much Na+ was translocated from root to shoot. K+ content was unchanged in C. paludosum and sea aster under saline conditions, but in safflower its uptake was inhibited by NaCl stress. Proline and sucrose contents of safflower and sea aster significantly increased by intensifying NaCl stress, furthermore in sea aster, glycinebetaine content significantly increased. Major contributions to decrease leaf osmotic potential by osmoregulation under NaCl stress were the accumulations of the compatible solutes (sucrose, proline, and glycinebetaine) in safflower. In C. paludosum and sea aster, an increase in ion concentrations (Na+ and Cl-) largely contributed to a decrease in leaf osmotic potential. This adaptation mechanism allows plants to keep a positive cell turgor by continuing water uptake so that seedlings can grow under salt stress.

The effects of NaCl stress on germination and early vegetative growth of 20 floricultural Asteraceae species and a wild halophyte species, sea aster (Aster tripolium L., Asteraceae) were studied to evaluate the possibility of selecting salt-tolerant species. Cluster analysis with the germination percentages relative to non-salinized control on the 10th day after sowing in 50 to 300 mM NaCl classified safflower (Carthamus tinctorius L. 'Roundish Leaf') as the most salt-tolerant. Although the germination percentage of sea aster decreased as salinity increased, its ability to germinate at 300 mM NaCl was next to that of safflower. To evaluate salt tolerance during the early vegetative growth of three species, sea aster, safflower, and Chrysanthemum paludosum Poir., plants were grown hydroponically at 25 and 50 mM NaCl for safflower and C. paludosum and at 150 and 300 mM NaCl for sea aster. Sea aster was much more tolerant of high salinity than were the other two species, and C. paludosum was more tolerant than was safflower. In conclusion, these three species differed in their sensitivities to salinity between germination and early vegetative growth. Therefore, selecting for salt-tolerant species is difficult because tolerance is genetically determined at the germination stage.

Cisplatin, cis-diamminedichloroplatinum(II) (CDDP) is one of the most important anticancer agents, initially producing good responses in various tumors. However, resistance to this drug often develops in various tumors, and additional administration decreases its chemotherapeutic efficacy. The precise mechanism of acquisition of resistance to this drug is still uncertain. However in the present study, we established two CDDP-resistant sublines A431/CDDP1 and A431/CDDP2 from human epidermoid carcinoma cell line A431. These resistant sublines were constituted by exposing A431 cells to a gradually increasing dose of CDDP (A431/CDDP1), and by mutagenic induction with mutagen (A431/CDDP2). A431/CDDP1 and A431/CDDP2 have developed 3.1 and 2.7 times more resistance to CDDP than the original A431 cell in terms of IC50. The two CDDP-resistant sublines showed cross-resistance to the CDDP analogue, carboplatin (CBDCA), but not to other chemotherapeutic drugs such as Adriamycin (ADR) and 5-fluorouracil (5-FU). These CDDP-resistant sublines were transplanted into nude mice to demonstrate the resistance to CDDP treatment in vivo. According to the in vitro assay, the mechanism of resistance in A431/CDDP1 and A431/CDDP2 seems to be based on a reduction of intracellular accumulation of CDDP, because their platinum concentration, which is the major component of CDDP, significantly declined. The established CDDP-resistant sublines may be used in further trials to improve the understanding of the mechanisms of resistance to CDDP.

A genomic clone encoding the precursor of a non-photosynthetic ferredoxin (Fd III) from maize has been isolated and characterized. In comparison with the corresponding cDNA, the gene (fedIII) was found to be interrupted in the 5'-untranslated region by an intron consisting of 3,037 bp. About 80% of the total region of this intron is organized into four classes of tandemly repeated sequences with monomeric lengths of about 200 bp, 320 bp, 130 bp, and 90 bp. This unique intron organization is present in the fedIII gene of three related maize cultivars examined.

It has been demonstrated that angiogenesis is required in the process of tumor progression and metastasis. Microvessel density (MVD) estimates tumor angiogenesis and is an independent indicator for predicting tumor metastasis in a variety of carcinomas. Platelet-derived endothelial cell growth factor (PD-ECGF) is known to be an angiogenic factor in vitro and in vivo. Of 55 patients with oral squamous cell carcinoma (OSCC), regional metastasis was absent in 35 and present in 20. Cases with lymph node metastasis showed significantly higher MVD (mean 61.0 +/- 28.8) than those without metastasis (mean 29.3 +/- 15.1; p0.001). A total of 37 cases (67.3%) were PD-ECGF-positive with a high MVD (mean 47.8 +/- 27.9) and 18 (32.7%) showed a negative PD-ECGF expression with a low MVD (mean 26.6 +/- 13.2). PD-ECGF expression was significantly correlated with the increment of MVD (p0.01). We suggest that MVD can be used as an independent prognostic indicator for predicting metastasis and that PD-ECGF activity plays an important role in the neovascularization of OSCC.

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