[Preparation and biological characteristics of extracellular matrix vesicle mimetics].

Objective: To investigate the characteristics of extracellular matrix vesicle mimetics prepared by mechanical extrusion and their effects on the cell viability and osteogenic differentiation potential of human periodontal ligament stem cells (PDLSC). Methods: PDLSC derived extracellular matrix vesicles were prepared by collagenase digestion, while the cell derived vesicle mimetics were simulated by mechanical extrusion. The obtained extracellular matrix vesicles and parental cell derived vesicle mimetics were divided into 4 groups: matrix vesicles derived from PDLSC cultured in basic medium for 7 days (PDLSC matrix vesicles, MVs), vesicle mimetics derived from PDLSC cultured in basic medium for 7 days (PDLSC vesicle mimetics, CVMs), matrix vesicles derived from PDLSC cultured in osteogenic inducing medium for 7 days (osteogenic-induced PDLSC matrix vesicles, O-MVs) and vesicle mimetics derived from PDLSC cultured in osteogenic inducing medium for 7 days (osteogenic-induced PDLSC vesicle mimetics, O-CVMs). Vesicles morphologies and sizes were observed by transmission electron microscopy and nanoparticle tracking analysis. Vesicles uptake was detected by immunofluorescence. With PDLSC as the control group, the effects of vesicles on the viability of PDLSC were detected by cell activity assay (cell counting kit-8), and the effects of vesicles on the osteogenic differentiation potential of PDLSC were detected by alizarin red staining and Western blotting. Results: Vesicles in MVs, O-MVs, CVMs and O-CVMs were all observed with a round structure (size 50-250 nm), and could be taken up by PDLSC without affecting the cell viability. Under osteogenic inducing conditions, PDLSC incubated with O-MVs or O-CVMs could produce more mineralized nodules than those in the control group (PDLSC). MVs, O-MVs, CVMs and O-CVMs could promote the expression of osteogenic-related proteins in PDLSC. PDLSC in group O-CVMs showed significant higher expressions of osteogenic-related proteins, including alkaline phosphatase (ALP) (1.571±0.348), osteopontin (OPN) (1.827±0.627) and osteocalcin (OCN) (1.798±0.537) compared to MVs (ALP: 1.156±0.170, OPN: 1.260±0.293, OCN: 1.286±0.302) (P<0.05). Compared to CMVs-incubated PDLSC, O-CVMs-incubated PDLSC expressed more Runt-related transcription factor 2 (1.632±0.455 vs 1.176±0.128) and OPN (1.827±0.627 vs 1.428±0.427) (P<0.05). Moreover, there was no significant difference in the expression levels of osteoblast-related proteins in PDLSC cultured with MVs, O-MVs and CVMs (P>0.05). Conclusions: The vesicle mimetics prepared by mechanical extrusion method are similar in shape and size to the extracellular matrix vesicles. MVs, O-MVs, CVMs and O-CVMs do not affect the cell viability of PDLSC, and can promote the osteogenic differentiation potential of PDLSC to a certain extent.

[Periodontal tissue regeneration: current therapeutic strategies and future directions in further research].

Chronic and progressive destruction/damage of the periodontal tissues resulted from periodontitis is the leading cause of tooth loss in adults. Traditional periodontal therapies such as scaling and root planning or flap surgery have demonstrated effective in controlling local inflammation and in suppressing/arresting the disease progression of periodontitis. However, those infection control measures cannot help to regenerate lost periodontal tissues to a statistically or clinically significant degree. Although some successes regarding the reduction of the intrabony defect and maintenance of the periodontal homeostasis have been achieved in periodontal regenerative procedures, comprising but not limited to guided tissue regeneration (GTR) or bone grafting technique, the restorative effectiveness of the architecture and function of the lost or injured tissues is far from our clinical expectation. The use of the concept, technique, and method of tissue engineering for periodontal regeneration is a hotspot and animal studies have shown interesting outcomes in terms of functional regeneration of lost/damaged support tissues in the periodontium, including alveolar bone, periodontal ligament, and cementum. However, numerous issues need to be addressed before those regenerative approaches can be responsibly transformed to novel clinical therapies. Recently, paradigm that induces homing of host stem cells to site of the periodontium and encourage the body's innate capability to repair is a new research field termed endogenous regeneration. Given that endogenous regenerative technique avoids ex-vivo cell culture and transplantation, it should be relatively easier to be used in the treatment of clinical patients. Due to the limited oral microenvironment and harsh periodontal local condition for tissue regeneration, as well as poor understanding of periodontal regenerative biology, there is still a long way ahead to explore new effective, practical, and economical therapies to save and protect natural tooth and for combating highly prevalent periodontal disease.

[Long non-coding RNA LINC01133 regulates cementogenic differentiation of human periodontal ligament stem cells by modulating mitochondrial functions].

Objective: To investigate the effects of long non-coding RNA (lncRNA) LINC01133 on the cementogenic differentiation of human periodontal ligament stem cells (hPDLSC) and the underlying mechanism. Methods: A total of 12 teeth were harvested from 10 patients aged 17-30 years in the Department of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military Medical University for impacted or orthodontic reasons from September 2021 to January 2022. The hPDLSCs were isolated from the teeth and transfected with small interfering RNA-LINC01133 (si-LINC01133) or small interfering RNA-negative control (si-NC). The si-LINC01133 was regarded as the experimental group, and the si-NC was regarded as the control one. The silencing efficiency of LINC01133 in the hPDLSCs was evaluated by real-time quantitative PCR (RT-qPCR). Western blotting was used to detect the protein expression levels of cementogenic differentiation-related factors including bone sialoprotein (BSP), cementum attachment protein (CAP), and cementum protein-1 (CEMP-1). Mitochondrial reactive oxygen species (mtROS) production was assessed using the MitoSox by flow cytometry. Mitochondrial membrane potential (MMP) was detected by JC-1 fluorescence staining. Mitochondrial respiratory chain complexes proteins including NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8 (NDUFB8), succinate dehydrogenase complex flavoprotein subunit A (SDHA), ubiquinol-cytochrome c reductase core protein 1 (UQCR1), cytochrome c oxidase subunit 4 isoform 1 (COXⅣ), and ATP synthase F1 subunit alpha (ATP5A) were evaluated by Western blotting. Results: The expression levels of LINC01133 could be suppressed by more than 60% with si-LINC01133 (control group: 1.000±0.000, experimental group: 0.385±0.128) (t=10.72, P<0.01). Suppression of LINC01133 in hPDLSCs decreased the levels of cementogenic differentiation-related proteins including BSP (control group: 1.000±0.000, experimental group: 0.664±0.179) (t=4.62, P<0.01) and CAP (control group: 1.000±0.000, experimental group: 0.736±0.229) (t=2.83, P<0.05). Suppression of LINC01133 in hPDLSCs increased the production of mtROS (control group: 1.000±0.000, experimental group: 1.458±0.185) (t=4.96, P<0.05) and the expression of NDUFB8 (control group: 1.000±0.000, experimental group: 1.683±0.397) (t=3.45, P<0.05), as well as decreased MMP levels (control group: 1.000±0.000, experimental group: 0.209±0.029) (t=53.99, P<0.01) and the expression of SDHA (control group: 1.000±0.000, experimental group: 0.428±0.228) (t=5.02, P<0.05). No significant changes in the UQCR1, COXⅣ, and ATP5A expression levels were found between the control group and exprimental group (P>0.05). Conclusions: LINC01133 regulates the cementogenic differentiation of hPDLSCs possibly via modulating the mitochondrial functions.

[Inflammatory factors in periodontitis patients and their effects toward the occurrence of gestational diabetes mellitus: a case-control study].

Objective: To explore the effects of periodontitis and inflammatory factors toward the occurrence of gestational diabetes mellitus (GDM). Methods: Pregnant women who came to the Department of Obstetrics, Northwest Women's and Children's Hospital for prenatal examinations during March to November of 2021 were invited to participate in this study. Participants with GDM who met the inclusion criteria (n=100) were assigned into the case group; while healthy participants (n=100) were assigned into the control group. Information of participants from the two groups were collected by questionnaires and periodontal statuses were clinically recorded in the meantime. Gingival crevicular fluid (GCF) and venous blood were also collected from participants of two groups to analyze the expression levels of inflammatory factors like C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, IL-8, IL-10 and IL-33. Factors different between the two groups were included in the multivariate regression analysis model to determine the risk factors of GDM. Results: The age of participants was (33.4±5.1) years in case group and (30.5±4.5) years in control group respectively, which had statistical differences (t=4.33,P<0.001). Besides, the body mass index of participants from case group was also significantly higher than control group [(28.11±3.85) kg/m2 vs. (23.31±3.15) kg/m2, t=9.65, P<0.001]. Participants with GDM had more adverse periodontal clinical parameters. Prevalence of periodontitis in GDM group was 47.0% (47/100) compared with 29.0% (29/100) in control group (χ²=6.88, P=0.009). Multivariate regression analysis results indicated that periodontitis was a critical risk factor for the occurrence of GDM (OR=1.882, P<0.001). Besides, GCF IL-8, serum TNF-α, IL-8 and IL-10 were also risk factors of GDM due to their higher expressions. Among them, TNF-α in serum (OR=2.077) and IL-8 in serum (OR=2.060) had more significant impacts (P<0.001). Conclusions: This study demonstrated that periodontitis was associated with the occurrence of GDM. Up-regulation of serum pro-inflammatory mediators leaded by local periodontal inflammatory microenvironment might play a critical role in this pathological process.

Leveraging Stem Cell Homing for Therapeutic Regeneration.

Resident stem cell pools in many tissues/organs are responsible not only for tissue maintenance during physiologic turnover but also for the process of wound repair following injury. With inspiration from stem cell trafficking within the body under physiologic and pathologic conditions, recent advances have been made toward inducing stem cell mobilization and directing patients' own cells to sites of interest for treating a broad spectrum of diseases. An evolving body of work corroborates that delivering guidance cues can mobilize stem cells from the bone marrow and drive these cells toward a specific region. In addition, the transplantation of cell-friendly biomaterials incorporating certain biomolecules has led to the regeneration of lost/damaged tissue without the need for delivering cellular materials manipulated ex vivo. Recently, cell homing has resulted in remarkable biological discoveries in the laboratory as well as great curative successes in preclinical scenarios. Here, we review the biological evidence underlying in vivo cell mobilization and homing with the aim of leveraging endogenous reparative cells for therapeutic applications. Considering both the promise and the obstacles of this approach, we discuss how matrix components of the in vivo milieu can be modified to promote the native regenerative process and inspire future tissue-engineering design.

[Periodontal tissue engineering and regeneration].

The clinical management of periodontal disease is a global concern, and the regeneration of periodontal tissue defects due to periodontitis faces a huge challenge in the field of regenerative dentistry. Although conventional periodontal therapies focusing on in flammation control could stop or delay the progression of the disease, periodontal regeneration remains an elusive but laudable goal. Since late 1980s, concerted efforts have been made to accelerate and augment periodontal repair by using guided tissue regeneration (GTR), guided bone regeneration (GBR) and a wide range of other regenerative paradigms. Those advances have largely improved the clinical outcomes of periodontal therapies. In the past several years of 21st century, many progresses were made in the developments of stem cell therapy and tissue engineering, including remarkable biological discoveries in the laboratory as well as great curative successes in preclinical scenarios. The use of the principles, techniques and procedures of tissue engineering in periodontology showed great potential to regenerate new functional periodontal tissues such as alveolar bone, periodontal ligament, root cementum and finally and predictably the normal structure and functionality of the periodontium around a previously diseased tooth.

[Effect of Tripterygium wilfordii polycoride upon inflammation and TLR4/MyD88 signaling pathway in ulcerative colitis rats model].

OBJECTIVE: To observe the effect of Tripterygium wilfordii polycoride (TWP) on ulcerative colitis (UC), and its intervention effect on toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling pathway, thus to investigate its possible mechanism. METHODS: Trinitrobenzene sulfonic acid (TNBS)/ethanol enema method was used to set up the UC rat model. With random number table, 90 male Wistar rats were divided into normal control group, model group, TWP low, medium and high dose group (3, 6, 12 mg/kg, respectively) and azathioprine (AZA) group (6 mg/kg), with 15 rates in each group. Four days after enema, rates in each group were given corresponding drug lavage for 14 consecutive days. Disease activity index (DAI), colon gross morphological damage and histological grading of each group were observed. Using Western blot and reverse transcription (RT)-PCR method, the TLR4/MyD88 signaling pathway-related proteins in UC rat intestinal tissue were detected, namely TLR4, MyD88, tumor necrosis factor receptor related factor 6 (TRAF-6), nuclear factor kappa B (NF-κB), tumor necrosis factor alpha (TNF-α), and interleukin-1 beta (IL-1ß). RESULTS: The DAI, colon gross morphological damage, and histological grading of the model group were significantly higher than that of the normal control group (all P<0.01), indicating successful establishment of UC model. The DAI, colon gross morphological damage and histological grading of the TWP high dose group were lower than those of the model group (0.87±0.25 vs 1.60±0.76, 3.93±1.94 vs 5.40±2.21, 5.45±2.73 vs 13.27±3.50, P<0.05). Compared with the normal control group, the mRNA and protein expressions of TLR4, MyD88, TRAF-6, NF-κB, TNF-α, and IL-1ß in the model group rats were significantly increased (all P<0.01); which were significantly decreased in the TWP high dose group compared with model group rats (mRNA: 2.166±0.475 vs 5.647±0.275, 1.295±0.087 vs 3.774±0.418, 1.125±0.188 vs 2.535±0.320, 1.201±0.152 vs 2.082±0.077, 1.525±0.218 vs 3.094±0.022, 1.797±0.257 vs 17.152±0.145; protein: 0.252±0.010 vs 0.277±0.008, 0.172±0.002 vs 0.213±0.005, 0.233±0.006 vs 0.248±0.003, 0.099±0.003 vs 0.122±0.007, 0.238±0.002 vs 0.252±0.005, 0.235±0.003 vs 0.245±0.006, all P<0.05), also decreased in the AZA group (all P<0.01); and there were no significant differences between the TWP high dose group and the AZA group (all P>0.05). CONCLUSIONS: TWP can alleviate intestinal inflammation, promote healing of mucosa, showing a therapeutic effect for UC. One of its mechanisms may be through inhibiting the expression of TLR4, affecting the expression of TRAF-6, which is downstream to MyD66 signaling pathway, thus to suppress the activation of NF-κB and reduce the release of inflammatory factor such as TNF-α and IL-1ß.

Differences between high- and low-motility buffalo sperm identified by comparative proteomics.

This study was undertaken to investigate differences in protein expression between high- and low-motility sperm of swamp buffalo. The research used two-dimensional gel electrophoresis (2DE) coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS) to analyse the different proteins. The results showed 18 different expression protein spots between high- and low-motility buffalo sperm; eight of these proteins were up-regulated in low-motility sperm, five were down-regulated, one deleted and four proteins specifically expressed. Finally, four proteins were successfully identified by MS as belonging to three unique proteins; they are outer dense fibre of sperm tails protein 2 (ODF2), ATP synthase subunit alpha (ATP5A1) and succinyl-CoA synthetase subunit beta (SUCLG2). In summary, these results help to develop an understanding of the molecular mechanisms associated with low-motility sperm and provide clues for finding molecular markers associated with sperm motility.

Expression of ß-glucuronidase on the surface of bacteria enhances activation of glucuronide prodrugs.

Extracellular activation of hydrophilic glucuronide prodrugs by ß-glucuronidase (ßG) was examined to increase the therapeutic efficacy of bacteria-directed enzyme prodrug therapy (BDEPT). ßG was expressed on the surface of Escherichia coli by fusion to either the bacterial autotransporter protein Adhesin (membrane ßG (mßG)/AIDA) or the lipoprotein (lpp) outermembrane protein A (mßG/lpp). Both mßG/AIDA and mßG/lpp were expressed on the bacterial surface, but only mßG/AIDA displayed enzymatic activity. The rate of substrate hydrolysis by mßG/AIDA-BL21cells was 2.6-fold greater than by pßG-BL21 cells, which express periplasmic ßG. Human colon cancer HCT116 cells that were incubated with mßG/AIDA-BL21 bacteria were sensitive to a glucuronide prodrug (p-hydroxy aniline mustard ß-D-glucuronide, HAMG) with an half maximal inhibitory concentration (IC50) value of 226.53±45.4 µM, similar to the IC50 value of the active drug (p-hydroxy aniline mustard, pHAM; 70.6±6.75 µM), indicating that mßG/AIDA on BL21 bacteria could rapidly and efficiently convert HAMG to an active anticancer agent. These results suggest that surface display of functional ßG on bacteria can enhance the hydrolysis of glucuronide prodrugs and may increase the effectiveness of BDEPT.

Laparoscopic Witzel gastrostomy--a reappraised technique.

BACKGROUND: Laparoscopic gastrostomy is the best alternative for long-term enteral feeding when percutaneous endoscopic gastrostomy is not possible. The aim of the present study was to determine the feasibility, complications, adequacy of feeding support, and tolerability of laparoscopic Witzel gastrostomy (LWG) in head and neck cancer patients. The initial results and the results of extended follow-up were evaluated. METHODS: A consecutive series of 48 patients with stenotic head and neck or esophageal cancer were referred for laparoscopic gastrostomy. The patients consisted of 42 men and 6 women aged 36 to 82 years (mean, 54 years). After laparoscopic placement of a Foley catheter of 16 F into the stomach, a seromuscular tunnel 4 cm in length is created, embedding the catheter by interrupted sutures. Three stay sutures for gastropexy are fixed and tied on the abdominal skin at the end of the procedure. The mean duration of the procedure was 62.4 +/- 11 min (52-124 min). RESULTS: Laparoscopic Witzel gastrostomy could be performed successfully in all patients with aerodigestive cancer. None of the laparoscopic gastrostomy tube placement procedures was converted to an open surgery, and none of the 48 patients in this series died as a result of the laparoscopic procedure. All LWG complications (11%) were minor, consisting of superficial wound infections, balloon rupture, and chronic granulation. No major complications were encountered. The mean usage time of gastrostomy was 6.3 +/- 5.3 months. CONCLUSIONS: Current techniques of LWG could be an alternative to percutaneous endoscopic gastrostomy (PEG) for long-term enteral access, because it has proved to be safe and reproducible with relatively few complications.

Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1.

Extracellular signal-regulated kinase activity is essential for mediating cell cycle progression from G(1) phase to S phase (DNA synthesis). In contrast, the role of extracellular signal-regulated kinase during G(2) phase and mitosis (M phase) is largely undefined. Previous studies have suggested that inhibition of basal extracellular signal-regulated kinase activity delays G(2)- and M-phase progression. In the current investigation, we have examined the consequence of activating the extracellular signal-regulated kinase pathway during G(2) phase on subsequent progression through mitosis. Using synchronized HeLa cells, we show that activation of the extracellular signal-regulated kinase pathway with phorbol 12-myristate 13-acetate or epidermal growth factor during G(2) phase causes a rapid cell cycle arrest in G(2) as measured by flow cytometry, mitotic indices and cyclin B1 expression. This G(2)-phase arrest was reversed by pre-treatment with bisindolylmaleimide or U0126, which are selective inhibitors of protein kinase C proteins or the extracellular signal-regulated kinase activators, MEK1/2, respectively. The extracellular signal-regulated kinase-mediated delay in M-phase entry appeared to involve de novo synthesis of the cyclin-dependent kinase inhibitor, p21(CIP1), during G(2) through a p53-independent mechanism. To establish a function for the increased expression of p21(CIP1) and delayed cell cycle progression, we show that extracellular signal-regulated kinase activation in G(2)-phase cells results in an increased number of cells containing chromosome aberrations characteristic of genomic instability. The presence of chromosome aberrations following extracellular signal-regulated kinase activation during G(2)-phase was further augmented in cells lacking p21(CIP1). These findings suggest that p21(CIP1) mediated inhibition of cell cycle progression during G(2)/M phase protects against inappropriate activation of signalling pathways, which may cause excessive chromosome damage and be detrimental to cell survival.

Molecular detection of disseminated tumor cells in the peripheral blood of patients with gastric cancer: evaluation of their prognostic significance.

Early detection of disseminated tumor cells in the peripheral blood of patients with early stage gastric cancer could help to improve the outcome after tumor resection. The aim of this study is to evaluate the prognostic significance of tumor-related mRNA for the detection of circulating tumor cells in gastric cancer patients by a reverse-transcriptase polymerase chain reaction (RT-PCR) method. We simultaneously analyzed human telomerase reverse transcriptase (hTERT), cytokeratin-19 (CK-19), cytokeratin-20 (CK-20) and carcinoembryonic antigen (CEA) mRNA (messenger RNA) expression in the peripheral blood of 42 gastric cancer patients and 30 healthy individuals. Additionally, analyses were carried out for the correlation of these four molecular markers with patients' clinicopathologic features, as well as the occurrence of postoperative recurrence/metastasis. Among 42 gastric cancer patients, the prevalence of mRNA for hTERT, CK-19, CK-20, and CEA was 61.9% (26/42), 69% (29/42), 61.9% (26/42), and 78.6% (33/42), respectively. All 30 healthy individuals were negative for hTERT and CEA mRNA, while two were positive for either CK-19 mRNA or CK-20 mRNA. Positive CEA mRNA was significantly correlated with tumor size p=0.008), vessel invasion (p=0.001), depth of tumor invasion (p=0.007), lymph node metastasis (p< 0.001), and TNM stage (p<0.001). In addition, the multivariate logistic regression demonstrated that CEA mRNA expression was an independent and significant predictor for postoperative recurrence/metastasis (p=0.032). Our findings suggest that CEA mRNA may be a more reliable marker than hTERT, CK-19 and CK-20 for the detection of circulating cancer cells in gastric cancer patients' peripheral blood. Patients with positive CEA mRNA expression in peripheral blood have a significantly higher risk of postoperative recurrence/metastasis.

Detection of circulating cancer cells with K-ras oncogene using membrane array.

K-ras oncogene is frequently found in human cancers and thus may serve as a potential diagnostic marker for cancer cells in circulation. So far, there is no reliable method for detecting cancer cells with K-ras oncogene in peripheral blood. The objective of this study was to develop a diagnostic membrane array using activated K-ras oncogene-associated molecules as detection targets. In our previous study, cDNA microarray analysis showed that there were 94 genes differentially expressed in K-ras mutant stably transfected adrenocortical cells. In the present study, we obtained 22 up-regulated genes in the closest relation to K-ras oncogene through bioinformatic analysis. At first, we carried out membrane array analysis by using in vitro culture cells. We demonstrated that this diagnostic technique was feasible and highly sensitive. A number as low as 5 cancer cells bearing K-ras oncogene in 1 ml of blood could be distinctively detected. Then, we collected blood specimens from 76 cancer patients. Direct sequencing analysis of these 76 samples showed that K-ras mutation was present in 43 patients with mutation sites mainly at codons 13, 15 and 61, which have been commonly established to be activated sites. We subsequently analyzed these 76 specimens with our diagnostic membrane array. Thirty-nine specimens were detected as positive for activated K-ras oncogene. Eighty percent (12/15) of mutations occurred at codon 13, 72.7% (8/11) at codon 61, and 88.9% (8/9) at codon 15 were accurately detected by our diagnostic membrane. Finally, through a series of biostatistical analyses, the sensitivity, specificity and accuracy of the diagnostic membrane array were 83.7, 90.9 and 86.8%, respectively. These findings suggest that the K-ras oncogene membrane array has a great potential for further investigation and clinical application.

Retinoblastoma protein (pRB) was significantly phosphorylated through a Ras-to-MAPK pathway in mutant K-ras stably transfected human adrenocortical cells.

Our previous studies have shown that the cell proliferation rate, mRNA levels of p450scc, p450c17, and 3betaHSD, and secretion of cortisol were significantly increased in human adrenocortical cells stably transfected with mutated K-ras expression plasmid "pK568MRSV" after being inducted with IPTG. In addition, the increased level was a time-dependent manner. However, the levels of p450, p450scc, p450c17, 3betaHSD, cortisol, and cell proliferation rate were inhibited by a MEK phospholation inhibitor, PD098059. The above results prove that mutated K-ras oncogene is able to regulate tumorigenesis and steroidogenesis through a Ras-RAF-MEK-MAPK signal transduction pathway. The aim of this study was to investigate regulated factors in this pathway and also examine whether the other signal transduction pathways or other moles involved in tumorigenesis or steroidogenesis. In the first year, we analyzed gene profiles of mutant K-ras-transfected adrenocortical cells by DNA microarray to determine the gene expression related to cell cycle, signal transduction, apoptosis, tumorigenesis, steroidogenesis, and other expressed sequence tag. After being affected by the K-ras mutant, gene expression was significantly increased in some upregulated genes. Human zinc-finger protein 22 increased by 28.5 times, Osteopontin increased by 5.8 times, LIM domain Kinase 2 (LIMK2) increased by 3.3 times, Homo sapiens dual-specificity tyrosine-(Y)-phosphorylation regulated Kinase 2 (DYRK2) increased by 2.2 times, and human syntaxin 3 increased by two times. On the other hand, significant decreases in gene expression were also observed in some downregulated genes. Retinoblastoma binding protein 1 (RBBP1) decreased by four times, Homo sapiens craniofacial development protein 1 (CFDP1) decreased by 2.4 times, DAP Kinase-related apoptosis-inducing protein Kinase 1 (DRAK1) decreased by 2.3 times, SKI-interacting protein (SKIP) decreased by 2.2 times, and human poly(A)-Binding protein (PABP) decreased by 2.1 times. In all significant differentially expressed genes, preliminary analysis by bioinformatics revealed that after induced K-ras mutant expression by isopropyl thiogalctoside (IPTG), the downregulation of RBBP1 gene was most correlated to cell proliferation. RBBP1 can bind with RB/E2F to form a mSIN3-HDAC complex, which induces cell cycle arrest in the G1/G0 stage by repressing transcription of E2F-regulated genes. The result of a Northern blot showed that RBBP1 were inhibited after an induction of IPTG for 36 h. Another Northern blot analysis proved that mRNA levels of cyclin D1 and c-myc increased in proportion to K-ras expression. Finally, Western blot was carried out, and the results showed that phosphorylated pRB also increased. Taken together, we infer that the mutant K-ras oncogene promoted the cells to proceed to the G1/S stage by the inhibiting the formation of RB/RBBP1-dependent repressor complex from binding with the SIN3-HDAC complex, which resulted in the acetylation of histone to active transcription of E2F-regulated genes. However, the roles of the other differentially expressed genes involved in cell proliferation, cell morphologic change, tumorigenesis, or steroidogenesis still need further investigation.

Actinomycin D binds strongly to d(TGTCATTG), a single-stranded DNA devoid of GpC sites.

Despite the absence of the GpC sequence and complete self-complementarity, d(CGTCGTCG) has recently been shown to bind strongly to actinomycin D (ACTD) with a binding density of about one drug molecule per strand. To further elucidate the nature of such a binding, studies are herein made with single-base G --> A and C --> T replacements in d(CGTCGTCG) to identify the DNA bases that play important roles in the strong ACTD binding of this oligomer. On the basis of these results, the octamer d(TGTCATTG) has been identified as a potentially strong ACTD binder. Indeed, binding titration confirms such an expectation and reveals an ACTD binding constant of about 1 x 10(7) M(-1) and a binding density of roughly 0.8 drug molecule per DNA strand for this strong binding mode. Similar binding studies with single-base substitutions on d(TGTCATTG) further reveal the relative importance of the C and G bases on its ACTD binding, with the 3'-terminus G appearing to be the most crucial base. Further base substitutions lead to the conclusion that these C and G bases act in concert rather than individually in the ACTD binding of d(TGTCATTG). Spectral comparisons with the apparently single-stranded GpC-containing d(TGCTTTG) led to the proposal of a speculated monomeric hairpin binding model to account for the experimental observations. This model makes use of the notion that ACTD prefers to have the 3'-sides of both G bases stacking on the opposite faces of its planar phenoxazone chromophore, a principle akin to its classic preference for the GpC sequence in duplex form. The finding that ACTD can bind strongly to single-stranded DNA of special sequence motifs may have important implications.

Actinomycin D binds strongly to d(CGACGACG) and d(CGTCGTCG).

Earlier calorimetric studies had indicated that despite the absence of a GpC sequence, the self-complementary octamer d(CGTCGACG) binds strongly to actinomycin D (ACTD) with high cooperativity and a 2:1 drug/duplex ratio. A subsequent optical spectral study with related oligomers led us to suggest that ACTD may likely stack at the G. C basepairs of the duplex termini. New findings are reported herein to indicate that despite the lack of complete self-complementarity, oligomers of d(CGXCGXCG) [X = A or T] motif exhibit unusually strong ACTD affinities with binding constants of roughly 2 x 10(7) M(-1) and binding densities of 1 drug molecule per strand. The ACTD binding affinity for the corresponding heteroduplex obtained by annealing these two oligomers is, however, considerably reduced. Although spectroscopic results with related oligomers obtained by removing, replacing, or appending bases at the termini appear to be consistent with the end-stacking model, capillary electrophoretic (CE) evidence provides additional insights into the binding mode. CE experiments with the self-complementary oligomers d(CGAGCTCG) and d(CGTCGACG) revealed contrasting migration patterns in the presence of ACTD, with mobility retardation and acceleration exhibited by the GpC- and non-GpC-containing octamers, respectively, whereas the X/X-mismatched d(CGXCGXCG) experienced retardation. These results, along with those of related oligomers, suggest that ACTD may in fact stack at the duplex stem end of a monomeric hairpin or at the 3'-end of dG as a single strand. The seemingly cooperative ACTD binding and the curved Scatchard plot for the self-complementary d(CGTCGACG) may thus be attributed to the drug-induced duplex denaturation resulting from strong binding to single strands of d(CGXCGYCG) motif. Detailed structural information on the ACTD-DNA complexes, however, must await further NMR investigations.

Influence of portal hypertension on secretion of gastric mucus in rats.

OBJECTIVE: To assess the effect of portal hypertension on gastric adherent and soluble mucus in rats. DESIGN: Experimental study. SETTING: Teaching hospital, Taiwan. MATERIAL: 30 male Wistar rats. INTERVENTIONS: Portal hypertension was induced experimentally by partial ligation of the portal vein in 20 male Wistar rats: ten rats were examined after 4 weeks and the remaining 10 after 8. Another group of 10 rats (controls) had sham operations. MAIN OUTCOME MEASURES: Portal pressure, the severity of gross gastric mucosal lesions, and measurement of gastric adherent and soluble mucus. RESULTS: The portal pressure and the gross mucosal damage differed significantly between the experimental and the control groups (p < 0.01). There was significantly less gastric adherent mucus in the two experimental groups than in the control group (p = 0.002 and <0.001, respectively), whereas there was no significant differences in the amount of gastric soluble mucus (p = 0.5 and 0.1, respectively). The reduction in the gastric adherent mucus was closely related to the increase in portal pressure (p < 0.001) and the severity of portal hypertension-induced gastropathy (p < 0.001). CONCLUSIONS: Gastric adherent mucus may have an important role in the pathogenesis of portal hypertensive gastropathy, and its protective capacity is reduced by portal hypertension, as indicated by the decrease in gastric adherent mucus.