Partial oxidation of dimethyl ether using the structured catalyst Rh/Al2O3/Al prepared through the anodic oxidation of aluminum.

The partial oxidation of dimethyl ether (DME) was investigated using the structured catalyst Rh/Al2O3/Al. The porous Al2O3 layer was synthesized on the aluminum plate through anodic oxidation in an oxalic-acid solution. It was observed that about 20 nm nanopores were well developed in the Al2O3 layer. The thickness of Al2O3 layer can be adjusted by controlling the anodizing time and current density. After pore-widening and hot-water treatment, the Al2O3/Al plate was calcined at 500 degrees C for 3 h. The obtained delta-Al2O3 had a specific surface area of 160 m2/g, making it fit to be used as a catalyst support. A microchannel reactor was designed and fabricated to evaluate the catalytic activity of Rh/Al2O3/Al in the partial oxidation of DME. The structured catalyst showed an 86% maximum hydrogen yield at 450 degrees C. On the other hand, the maximum syngas yield by a pack-bed-type catalyst could be attained by using a more than fivefold Rh amount compared to that used in the structured Rh/Al2O3/Al catalyst.

Effects of a combined alendronate and calcitriol agent (Maxmarvil) on bone metabolism in Korean postmenopausal women: a multicenter, double-blind, randomized, placebo-controlled study.

INTRODUCTION: A randomized, double-blind, prospective, 24-week clinical trial was performed to evaluate the effects of a combinative agent, Maxmarvil, of calcitriol (0.5 mug) and alendronate (5 mg) on bone metabolism in postmenopausal women. METHODS: A total of 217 postmenopausal women with osteoporosis were enrolled; 199 patients were randomly assigned to one of two treatment groups (Maxmarvil group or alfacalcidol group). None of the patients were vitamin-D-deficient, as assessed by serum 25-hydroxyvitamin D (25(OH)D), nor had they received any drugs affecting bone metabolism before enrollment. Bone mineral densities (BMD) of L1-L4 and the femur were measured by dual-energy X-ray absorptiometry (DXA) at the initial assessment and after 6 months of treatment. Serum biochemical assays, including serum calcium, 24-h urinary calcium excretion, and bone turnover markers (both bone-specific alkaline phosphatase [bsALP] and urine N-telopeptide [NTx]), were performed at the baseline and after 3 and 6 months of treatment. RESULTS: In the Maxmarvil group, the BMD of the lumbar spine increased up to 2.42+/-0.5% from the baseline after 6 months (p<0.05). On the other hand, the change in BMD in the alfacalcidol group was 0.28+/-0.5% after 6 months. There was no significant difference in femoral BMD between the two groups. The levels of bsALP and NTx were significantly lower in the Maxmarvil group than in the alfacalcidol group (-22.04+/-3.9% vs. -11.42+/-2.8% [p<0.05] and -25.46+/-5.2% vs. 1.24+/-6.2% [p<0.001], respectively). Interestingly, there was a significantly smaller amount of 24-h urinary calcium in the Maxmarvil group (p<0.05). CONCLUSIONS: Our study demonstrates that a combination of calcitriol and alendronate is quite effective in preventing bone loss, with the advantage of lesser hypercalciuric effect of calcitriol in the postmenopausal osteoporotic women.

Promoter CpG hypermethylation and downregulation of XAF1 expression in human urogenital malignancies: implication for attenuated p53 response to apoptotic stresses.

XIAP-associated factor 1 (XAF1) is a new candidate tumor suppressor, which has been known to exert proapoptotic effects by interfering with the caspase-inhibiting activity of XIAP. To explore the XAF1's candidacy for a suppressor in urogenital tumorigenesis, we investigated the XAF1 status in a series of cancer cell lines and primary tumors derived from the bladder, kidney and prostate. Expression of XAF1 transcript was undetectable or extremely low in 60% (3/5) of bladder, 66% (10/15) of kidney, and 100% (3/3) prostate cancer cell lines. Abnormal reduction of XAF1 was also found in 33% (18/55) of primary bladder and 40% (8/20) of primary kidney tumors, and showed a correlation with advanced stage and high grade of bladder tumor. Hypermethylation at 14 CpG sites in the 5' proximal region of the XAF1 promoter was highly prevalent in cancers versus adjacent normal or benign tissues and tightly associated with reduced gene expression. XAF1 expression enhanced the apoptotic response of tumor cells to chemotherapeutic agents, such as etoposide or 5-FU. While XAF1 expression did not influence the subcellular distribution or expression of XIAP, it elevated the protein stability of p53 and its target gene expression. Moreover, the apoptosis-sensitizing and growth suppression function of XAF1 was markedly impeded by blockade of p53 function. Collectively, our study demonstrates that epigenetic alteration of XAF1 is frequent in human urogenital cancers and may contribute to the malignant progression of tumors by rendering tumor cells a survival advantage partially through the attenuated p53 response to apoptotic stresses.

Expression and mutation analyses of MKK4, a candidate tumour suppressor gene encoded by chromosome 17p, in human gastric adenocarcinoma.

Homozygous deletion or somatic mutations of mitogen-activated protein kinase kinase 4 (MKK4), a candidate tumour suppressor gene located at 17p11, have been observed in many types of human tumours. To explore the likelihood that MKK4 acts as a suppressor in gastric tumorigenesis, we examined the expression and mutation status of MKK4 in 144 gastric tissues and cell line specimens. Expression of the MKK4 transcript was easily detectable in all normal and benign tumour tissues and none of 102 primary carcinomas and cell lines showed an abnormal reduction in MKK4 expression. Expression levels of MKK4 transcript showed no cancer-specific reduction in 43 matched sets and did not correlate with stage, grade and histopathological types of the tumours. Western blot analysis also revealed that MKK4 protein expression in carcinoma tissues and cell lines is comparable to non-cancerous tissues. A significant loss of heterozygosity (LOH) was detected at telomeric markers of the MKK4, locus. However, no allelic deletion of the MKK4 gene or at the centromeric loci was identified. Moreover, no evidences for somatic mutations leading to amino acid substitutions or frameshifts of MKK4 were identified in the carcinoma tissues and cell lines, whereas a substantial fraction of the same set showed allelic loss or mutations of the TP53 gene located at 17p13, suggesting that LOH at telomeric loci or the TP53 locus might not extend into the MKK4 gene in gastric cancers. In this study, we also report the identification of a highly conserved MKK4 processed pseudogene, which shares 95% homology with the coding region of the functional MKK4 transcript. Collectively, our data demonstrate that genomic deletion or somatic mutation of MKK4 is infrequent in gastric cancers, suggesting that MKK4 might not be a critical target of genetic inactivation in gastric tumorigenesis.

Enhanced IL-18 expression in common skin tumors.

Interleukin-18 (IL-18) has been found to have multiple effects upon various cells involved in inflammatory response. Recently we reported that B16 murine melanoma cells are able to produce IL-18, which is involved in the regulation of intracellular reactive oxygen intermediates (ROI) and Fas-ligand expression, indicating that IL-18 plays key role in the tumor activity of melanoma. In this study, we investigated the pattern of IL-18 expression in the human system. IL-18 production was tested by enzyme linked immunosorbent assay (ELISA) assay in various tumor cell lines, including Raji (Burkitt's lymphoma), IM-9 (B lymphoblast), Jurkat (acute T cell leukemia), SK-MES-1 (squamous cell carcinoma (SCC) cell line), SK-MEL-2, G-361, DM-4, and DX-3 (melanoma cell lines). ELISA tests showed that IL-18 was highly expressed in malignant skin tumors such as SK-MES-1, SK-MEL-2, G-361, DM-4, and DX-3 cell lines, thus suggesting that IL-18 production may be associated with the malignancy of skin tumors. Here, we report that enhanced IL-18 expression is positively correlated with malignant skin tumors such as SCC and melanoma, suggesting the importance role of IL-18 in malignancy of skin tumors. Taken together, expression of IL-18 by tumor cells in human skin tissue may provide an important clue to understand the pathogenesis of malignant skin tumors.

Frequent epigenetic inactivation of RASSF1A by aberrant promoter hypermethylation in human gastric adenocarcinoma.

Methylation associated inactivation of RASSF1, a putative tumor suppressor identified at 3p21.3, has been frequently observed in several human malignancies, including lung and breast cancers. To explore the penetrance of RASSF1 in gastric carcinogenesis, we performed expression and mutation analyses of 3 isotypes of RASSF1 (A, B, and C) in 150 gastric specimens, including 15 carcinoma cell lines. RASSF1A and RASSF1B transcripts were not expressed in 60% (9 of 15) and 33% (5 of 15) of gastric carcinoma cell lines, respectively, whereas RASSF1C was detectable in all cell lines. Bisulfite DNA sequencing analysis revealed that the CpG island in the RASSF1A promoter is hypermethylated in all RASSF1A-nonexpressing cell lines. In addition, both RASSF1A and RASSF1B were re-expressed by treatment with the demethylating agent 5-aza-2'-deoxycytidine. Among 90 primary gastric adenocarcinomas examined, 41 (46%) and 19 (21%) expressed no or abnormally low levels of RASSF1A and RASSF1B, respectively, and 12 (13%) tumors showed no expression of both isoforms. Loss or abnormal down-regulation of RASSF1A correlated with tumor stage and grade but not with histological types of tumors. Methylation-specific PCR analysis demonstrated that 95% (39 of 41) of RASSF1A-nonexpressing primary tumors are methylated at the CpG sites in the promoter, whereas none of the adjacent noncancerous or normal tissues are methylated. No somatic mutations were detected in RASSF1 transcripts expressed in unmethylated tumors. However, 10 methylated tumors, including 4 cell lines, showed low genomic levels of RASSF1 and expressed no RASSF1A transcripts, suggesting that RASSF1A inactivation might be caused by both epigenetic and genetic mechanisms in a subset of gastric adenocarcinomas. In conclusion, our data indicate that epigenetic transcriptional silencing of RASSF1, especially RASSF1A isoform, is a frequent event in gastric tumorigenesis and might play an important role in the malignant progression of gastric adenocarcinomas.

Frequent epigenetic inactivation of RASSF1A in human bladder carcinoma.

Allelic deletion or transcriptional silencing of RASSF1, a putative tumor suppressor at 3p21.3, has been found in a considerable proportion of lung, breast, and ovarian cancers. In this study, we analyzed the expression and mutation status of three RASSF1 isoforms (-A, -B, and -C) in 55 primary bladder carcinomas and 10 bladder and prostate cancer cell lines. The RASSF1A transcript was not found in 80% (4 of 5) and 100% (4 of 4) of bladder and prostate cell lines, respectively. Compared with normal bladder tissues, loss or significant reduction of RASSF1A was identified in 62% (34 of 55) of primary bladder carcinomas and 10 (83%) of 12 matched sets showed tumor-specific alteration of RASSF1A expression. Moreover, loss or abnormal down-regulation of RASSF1A correlated with advanced tumor stage. RASSF1B was undetectable in 60% (3 of 5) of bladder cell lines and in 31% (17 of 55) of primary tumors, but none of these tumors showed altered expression exclusively in RASSF1B. RASSF1C transcript was detected in all cell lines and primary tumors we examined. Expression of RASSF1A and RASSF1B was reactivated in all nonexpressor cell lines by treatment with the demethylating agent 5-aza-2'-deoxycytidine. Bisulfite DNA sequencing analysis revealed that aberrant hypermethylation at the CpG island in the RASSF1A promoter is strongly associated with the loss of RASSF1A expression in cell lines and uncultured primary tumors. Methylation-specific PCR and BstUI digestion analyses also demonstrated that 97% (33 of 34) of RASSF1A-nonexpressing primary tumors are methylated. Although somatic mutations were not identified in RASSF1 transcripts expressed in unmethylated tumors, 24% (9 of 37) of methylated cell lines and primary tumors showed detectable reductions in genomic levels of RASSF1, suggesting that RASSF1A inactivation might be caused by both epigenetic and genetic mechanisms in a subset of bladder tumors. Together, our data suggest that RASSF1A inactivation may play a critical role in the malignant progression of human bladder carcinomas.

Codon 54 polymorphism of the fatty acid binding protein 2 gene is associated with increased fat oxidation and hyperinsulinemia, but not with intestinal fatty acid absorption in Korean men.

The alanine to threonine substitution at codon 54 (Ala54Thr) of the fatty acid binding protein 2 (FABP2) gene has been reported to be associated with increased fat oxidation and insulin resistance in several populations. It has been hypothesized that Ala54Thr substitution results in enhanced intestinal uptake of fatty acids and thereby an impairment of insulin action, but this hypothesis has not been proven in vivo. We studied the association between the Ala54Thr polymorphism of the FABP2 gene and intestinal (3)H-oleic acid absorption, as well as basal insulin level, basal metabolic rate, and fat oxidation rate in 96 healthy young Korean men. Among our subjects, the allele frequency of the Ala54Thr substitution was 0.34. Subjects with Thr54-encoding allele were found to have a higher mean fasting plasma insulin concentration and a higher basal fat oxidation rate compared with the subjects who were homozygous for the Ala54-encoding allele. However, there was no significant difference in basal metabolic rate or (3)H-oleic acid absorption according to the FABP2 gene polymorphism. These results suggest that the Ala54Thr substitution in the FABP2 gene is associated with increased fat oxidation and hyperinsulinemia in normal Korean men, but these effects are not mediated by an increase in the intestinal fatty acid absorption.

Localization of the IGF binding domain and evaluation of the role of cysteine residues in IGF binding in IGF binding protein-4.

Our previous findings suggest that binding of IGF binding protein-4 (IGFBP-4) to IGFs is essential for the inhibitory effect of IGFBP-4 on the activity of IGFs, both in vitro and in vivo. Therefore, understanding the structural determinants of IGF binding in IGFBP-4 is important to the general understanding of the biology of the IGF system. This study sought to further localize the IGF binding domain and to evaluate the role of Cys residues in IGF binding. Our data revealed that full-length IGFBP-4 peptides lacking the residues Leu(72)-Ser(91) or Leu(72)-His(74) or Gly(75)-Ser(91) failed to bind to IGF-I or IGF-II, whereas deletion of the residue Leu(72) or residues Met(80)-Ser(91) led to a 2- to 3-fold reduction in IGF-I and IGF-II binding activity. The IGF-I and IGF-II binding activities were dramatically reduced by the single mutation, Cys9/Arg (>25-fold), and to a lesser degree, by the single mutation, Cys12/Arg (the first N-terminal Cys residue was designated Cys1). The mutation Cys17/Ser or Cys18/Tyr or Cys20/Ser each resulted in a similar but moderate ( approximately 5-fold) reduction in IGF-II binding activity. The IGF-I binding activity was also dramatically reduced by the mutation Cys18/Tyr, and to a lesser extent, by the mutation Cys17/Ser or Cys20/Ser. These data suggest: 1) the IGF-I and IGF-II binding domain in IGFBP-4 involves a hydrophobic motif (Leu(72)-Met(80)) located in the distal part of the conserved N-terminal region, and 2) the N-terminal Cys residues (Cys9 and Cys12) are more critical than the C-terminal Cys residues (Cys17 and Cys20) in affecting the IGF-I and IGF-II binding. Based on these data, we speculate that the structural determinants of IGF-I and IGF-II binding in IGFBP-4 are very similar, if not identical.

Temporary tattooing followed by Q-switched alexandrite laser for treatment of syringomas.

BACKGROUND: Syringomas usually develop in women as multiple skin-colored papules primarily seen on the periocular regions and cheeks. They can cause cosmetic problems and lead to poor self-esteem. Though several treatment modalities have been established, such as excision, electro/cryosurgery, chemical peeling, and CO2 laser surgery, none of them are satisfactory due to their limitations and side effects, for example, pain, prolonged healing time, postoperative erythema/pigmentary changes, and scarring. OBJECTIVE: The objective of this study was to develop a new treatment method for syringoma and to minimize the side effects through selective destruction of the tumor. METHODS: Six patients with multiple periorbital syringomas were enrolled in this study. The surface epithelium of the syringomas was vaporized by CO2 laser, and black ink was introduced in order to allow penetration to the dermis using iontophoresis. Subsequently the artificial tattoos were removed by Q-switched alexandrite laser. The results were evaluated clinically by both physicians and patients at 1, 2, 4, and 8 weeks after treatment. RESULTS: The majority of syringoma in the six patients disappeared by the first follow-up 1 week after treatment. There were no cases of prolonged erythema persisting beyond 2 weeks. Additional treatment was repeated in the same manner in order to remove the remaining syringomas in one patient. There were no recurrences during the 8-week follow-up period. CONCLUSION: Our new treatment was safer, less painful, nonscarring, and there was a quicker recovery period and less of a burden to repeat treatment when necessary.

Pregnancy-associated plasma protein-A accounts for the insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) proteolytic activity in human pregnancy serum and enhances the mitogenic activity of IGF by degrading IGFBP-4 in vitro.

Pregnancy-associated plasma protein-A (PAPP-A) has been identified as the insulin-like growth factor (IGF)-dependent IGF-binding protein-4 (IGFBP-4) protease produced by human fibroblasts. Recently, we found that serum proteases induced during human pregnancy cleaved IGFBP-4 in both an IGF-II-dependent and an IGF-II-independent fashion. This study sought to determine whether PAPP-A is the predominant IGFBP-4 protease in human pregnancy serum (PS) and to assess the in vitro role of serum PAPP-A. Immunoprecipitation with PAPP-A antibody effectively depleted PAPP-A from the PS and completely abolished both IGF-II-dependent and IGF-II-independent IGFBP-4 proteolytic activity in PS. Direct addition of PAPP-A antibody to PS completely blocked IGFBP-4 proteolysis and partially blocked IGFBP-5 proteolysis, but had no effect on IGFBP-3 proteolysis. To evaluate the role of serum PAPP-A, we tested whether PAPP-A in PS modulated the inhibitory activity of IGFBP-4 on IGF-II-induced cell proliferation in human osteosarcoma MG63 cells. The wild-type IGFBP-4 (WTBP-4; 200 ng/mL) failed to inhibit proliferation of the cells treated with PS (0.1% or 0.3%) alone or in combination with IGF-II (40 ng/mL), whereas the inhibitory effect of WTBP-4 was observed in the cells treated with nonpregnancy serum alone or in combination with IGF-II (P < 0.05). In contrast to WTBP-4, a protease-resistant IGFBP-4 was able to inhibit proliferation of the cells treated with PS alone or in combination with IGF-II (P < 0.05). In the presence of PAPP-A neutralizing antibody, the inhibitory effect of WTBP-4 on proliferation of the cells treated with IGF-II and PS was restored. In summary, these data demonstrate 1) that PAPP-A represents the predominant IGFBP-4 protease in PS; 2) that PAPP-A may in part contribute to IGFBP-5, but not IGFBP-3, proteolytic activity in PS; and 3) that PAPP-A enhances the bioactivity of IGFs in vitro by degrading IGFBP-4.

Effects of substance P and vasoactive intestinal peptide on interferon-gamma and interleukin-4 production in severe atopic dermatitis.

BACKGROUND: Recent studies have demonstrated that two T cell-derived lymphokines, interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), play a crucial role in the pathogenesis of atopic dermatitis (AD). It is known that neuropeptides, such as substance P (SP) and vasoactive intestinal peptide (VIP), have various immunomodulatory effects. Elevated levels of these neuropeptides and increased staining of SP positive nerve fibers have been reported in AD patients. OBJECTIVE: The study was designed to examine the effects of SP and VIP on the production of IFN-gamma and IL-4. The aim of the study was to establish whether these neuropeptides acted to affect cytokine release in the peripheral blood mononuclear cells (PBMCs) of AD patients. METHODS: The effects of SP and VIP on the production of IFN-gamma and IL-4 in phytohemagglutinin stimulated PBMC cultures over a 48-hour period were analyzed by enzyme-linked immunosorbent assay in 15 AD patients. Non-atopic individuals were used as a control group. RESULTS: Base cytokine profiles of AD patients showed significantly decreased IFN-gamma and increased IL-4 when weighed against non-atopic controls. Compared with controls, SP had a significant percentage enhancing effect on both IFN-gamma and IL-4 production at concentrations of 10(-8) M and 10(-6) M, however, this IFN-gamma up-regulatory effect of SP was reversed by spantide, a SP antagonist. The ratios of IFN-gamma: IL-4 production were significantly elevated in the SP treated AD group. Although VIP had no specific noticeable effects on the IFN-gamma and IL-4 production. CONCLUSIONS: Our data may suggest that SP has an influence on the immunomodulation of AD patient by regulating IFN-gamma production, either directly or indirectly. Vasoactive intestinal peptide, on the other hand, has no modulatory effects on the cytokine production of AD patients.

Mitogenic conversion of transforming growth factor-beta1 effect by oncogenic Ha-Ras-induced activation of the mitogen-activated protein kinase signaling pathway in human prostate cancer.

Elevated expression of transforming growth factor (TGF)-beta1 has been implicated in prostate tumorigenesis despite its growth-inhibitory effect on normal epithelial and carcinoma cells of the prostate. In this study, we identified that G1-to-S transition of the cell cycle is stimulated by TGF-beta1 in the prostate cancer cell line TSU-Pr1. No mutation of signal mediators, including Smads, and induction of PAI-1 transcription indicated that the TGF-beta1 signaling cascade is functionally intact in this cell line. Whereas pharmacological inhibitors of various mitogenic signaling pathways showed no effects, blockade of the mitogen-activated protein kinase (MAPK) pathway by the MAPK kinase 1 inhibitor PD98059 restored the growth inhibitory role of TGF-beta1 in TSU-Pr1, which carries an oncogenic mutation in Ha-Ras (V12). Moreover, expression of antisense Ha-Ras or dominant negative Raf-1 abrogated the mitogenic effect of TGF-beta1 in TSU-Pr1, and the TGF-beta1 inhibition of DU145 was switched to stimulation by V12Ha-Ras transfection. Whereas the negative growth regulation by TGF-beta1 was completely inhibited by dominant negative Smad2, Smad3, or Smad4, its mitogenic effect was not affected, suggesting that this action is Smad-independent. Interestingly, whereas the TGF-beta1-mediated up-regulation of p15INK4B and p21WAF1 transcription was abolished in TSU-Pr1 and V12Ha-Ras-transfected DU145, inhibition of the Ras/MAPK pathway restored the TGF-beta1 induction of these genes. Taken together, our data suggest that prostate carcinomas with the Ras/MAPK pathway activation might have a selective growth advantage by autocrine TGF-beta1 production.

Spontaneous expression of mRNA for IL-10, GM-CSF, TGF-beta, TGF-alpha, and IL-6 in peripheral blood mononuclear cells from atopic dermatitis.

BACKGROUND: Monocytes and T helper cells play major roles in the immunologic dysfunction of atopic dermatitis (AD). There have been many studies on the cytokine pattern to evaluate abnormalities of immune cells in AD, but the results were conflicting and most of these previous reports were performed with various mitogen-stimulation. OBJECTIVE: The purpose was to investigate de novo cytokine pattern in AD peripheral blood mononuclear cells (PBMC). We focused on the expression of cytokines that have effects on monocytes and T cells. METHODS: We measured mRNA expression of IL-10, GM-CSF, TGF-beta, TNF-alpha, and IL-6 in freshly isolated PBMC with semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The intensity of cytokine cDNA were normalized to that of beta-actin product as a standard marker. RESULTS: Interleukin-10 mRNA expression was significantly enhanced in AD compared with control subjects (P < .05). Spontaneous mRNA expression of TGF-beta and TNF-alpha was significantly lower in AD patients (P < .01). The level of GM-CSF mRNA expression was heterogenous and spontaneous mRNA expression was slightly increased in AD although the difference didn't reach the statistical significance. Interleukin-6 mRNA was not detected in most of AD and controls. CONCLUSION: Our data could represent in vivo cytokine expression state associated with monocytes and other immune cells. Increased expression of IL-10 and GM-CSF may be associated with monocyte dysfunction in AD although increase in the expression of GM-CSF mRNA was not statistically significant. Inhibitory effect of increased IL-10 was suggested on decreased expression of TNF-alpha mRNA. The role of TGF-beta in AD remains to be seen.

Loss of imprinting and elevated expression of wild-type p73 in human gastric adenocarcinoma.

The p73 gene located at 1p36.3 encodes for a protein with significant similarity to p53. To investigate the penetrance of p73 in gastric carcinogenesis, we analyzed the expression, allelotype, and mutation of p73 in five cell lines and 75 tissues. Although extremely low levels of p73 expression were observed in all noncancerous gastric tissues and four of five cell lines, a significant elevation of p73 was detected in 37 of 39 (94.9%) carcinoma tissues. Furthermore, a tumor-specific increase of p73 was identified in 14 of 16 (87.5%) matched sets. Allelotyping analysis using a StyI or BanI polymorphism revealed that 5 of 21 (23.8%) informative carcinomas, but none of 19 noncancerous cases, express p73 biallelically, suggesting the transcriptional activation of a silent allele in a subset of cancers. Whereas the transcription of an active allele was markedly induced by serum starvation or clump formation of the cells, treatment with 5-aza-2'deoxycytidine activated a silent allele with a subsequent up-regulation of an active allele, supporting the genomic imprinting and autoregulation of the gene. Allelic deletion or mutation of the gene was not found, and no association of p73 expression with the mutational status of p53 or expression of p21Waf1 was recognized. Taken together, this study argues that p73 is not a target of genetic alteration in gastric carcinogenesis and suggests that overexpression of p73 might be triggered by physiological stresses accompanied with outgrowth of tumors, such as hypoxia or nutrient deprivation.

Studies on the role of human insulin-like growth factor-II (IGF-II)-dependent IGF binding protein (hIGFBP)-4 protease in human osteoblasts using protease-resistant IGFBP-4 analogs.

To characterize the insulin-like growth factor binding protein-4 (IGFBP-4) protease produced by human osteoblasts (hOBs), we localized and determined the role of the proteolytic domains in human IGFBP-4 (hIGFBP-4) in modulating IGF-II actions. N-terminal amino acid sequence and mass spectrometric analyses of the 6xHis-tagged IGFBP-4 proteolytic fragments revealed that Met135-Lys136 was the only cleavage site recognized by the IGF-II-dependent IGFBP-4 protease produced by hOBs. This cleavage site was confirmed by the finding that deletion of His121 to Pro141 blocked proteolysis. However, unexpectedly, deletion of Pro94 to Gln119 containing no cleavage site had no effect on IGF-II binding activity but blocked proteolysis. Addition of the synthetic peptide corresponding to this region at concentrations of 250 or 1000 molar excess failed to block IGFBP-4 proteolysis. These data suggest that residues 94-119 may be involved in maintaining the IGFBP-4 conformation required to expose the cleavage site rather than being involved in direct protease-substrate binding. To determine the physiological significance of the IGF-II-dependent IGFBP-4 protease, we compared the effect of the wild-type IGFBP-4 and the protease-resistant IGFBP-4 analogs in blocking IGF-II-induced cell proliferation in normal hOBs, which produce IGFBP-4 protease, and MG63 cells, which do not produce IGFBP-4 protease. It was found that protease-resistant IGFBP-4 analogs were more potent than the wild-type protein in inhibiting IGF-II-induced cell proliferation in hOBs but not in MG63 cells. These data suggest that IGFBP-4 proteolytic fragments are not biologically active and that IGFBP-4 protease plays an important role in regulating IGFBP-4 bioavailability and consequently the mitogenic activity of IGFs in hOBs.

Expression of T cell receptor V beta chain in lesional skin of atopic dermatitis.

Atopic dermatitis is a chronic, relapsing inflammatory skin disorder characterized by local infiltration of T cells. To date, numerous reports have shown that Staphylococcus aureus may exacerbate atopic dermatitis, and superantigens produced by this organism are thought to be one of the major causative factors in atopic dermatitis. The purpose of this study was to evaluate the role of staphylococcal superantigen in atopic dermatitis by observing expression of the variable region of beta chain of T cell receptor (TCR V beta) in the inflammatory cells infiltrating cutaneous lesions of atopic dermatitis. Fourteen patients with atopic dermatitis were enrolled. Punch biopsy specimens were obtained from lesional and normal-appearing skin of all patients. The expression of TCR V beta was studied by means of immunohistochemical technique using monoclonal antibodies. In 4 out of 14 patients, the tendencies of preferential expression of specific TCR V beta were found in lesional skin. This study suggested that staphylococcal superantigen and its corresponding T cell subsets may act as causative or pathogenic factors in a subgroup of atopic dermatitis.

CD31 (PECAM-1) is a differentiation antigen lost during human CD4 T-cell maturation into Th1 or Th2 effector cells.

The CD31 antigen (PECAM-1) has been reported to be a stable marker for a human CD4 T-cell subpopulation unable to produce interleukin-4 (IL-4). We show here that CD31 expression is not stable inasmuch as CD4 T-cell lines and clones derived from cell-sorted neonatal CD31+ cells lose CD31 upon repetitive cycles of stimulation and IL-2 expansion. Moreover, various cytokines (IL-1 alpha, IL-4, IL-6, transforming growth factor-beta) fail to reinduce CD31 on CD31- clones. Whereas all CD31+ CD4 T cells rapidly express high levels of the CD45RO antigen and down-regulate the L-selectin antigen after priming, CD31 disappears more slowly because only part of the cells lose CD31 expression upon each cycle of stimulation. Loss of CD31 reflects a functional maturation of CD45RO+ cells since, in a system which favours the development of Th2 effectors, IL-4 is produced by CD31- but not CD31+ effector T cells, whereas interferon-gamma is produced by both types of cells. However, CD31 is not a Th1 marker since it is not expressed on several Th1 antigen-specific clones. We conclude that CD31 is a maturation marker expressed on the great majority of naive CD45RO- CD4 T cells and on a subset of CD45RO+ CD4 T cells that are at an intermediate stage of maturation.