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Cognitive behavioral therapy (CBT) is effective for headache disorders. However, it is unclear whether the emerging digital CBT is noninferior to face-to-face CBT. An indirect treatment comparison (ITC) meta-analysis was conducted to assess the relative effects between them using standard mean differences (SMDs). Effective sample size (ESS) and required sample size (RSS) were calculated to demonstrate the robustness of the results. Our study found that digital CBT had a similar effect on headache frequency reduction (SMD, 0.12; 95%CI, -2.45 to 2.63) compared with face-to-face CBT. The ESS had 84 participants, while the RSS had 466 participants to achieve the same power as a non-inferior head-to-head trial. Digital CBT is as effective as face-to-face CBT in preventing headache disorders. Due to the heterogeneity (I 2  = 94.5%, τ 2  = 1.83) and the fact that most of the included studies were on migraine prevention, further head-to-head trials are warranted., (© 2024. The Author(s).)

Dietary trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) is a potential candidate in anti-obesity trials. A transgenic mouse was previously successfully established to determine the anti-obesity properties of t10c12-CLA in male mice that could produce endogenous t10c12-CLA. To test whether there is a different impact of t10c12-CLA on lipid metabolism in both sexes, this study investigated the adiposity and metabolic profiles of female Pai mice that exhibited a dose-dependent expression of foreign Pai gene and a shift of t10c12-CLA content in tested tissues. Compared to their gender-match wild-type littermates, Pai mice had no fat reduction but exhibited enhanced lipolysis and thermogenesis by phosphorylated hormone-sensitive lipase and up-regulating uncoupling proteins in brown adipose tissue. Simultaneously, Pai mice showed hepatic steatosis and hypertriglyceridemia by decreasing gene expression involved in lipid and glucose metabolism. Further investigations revealed that t10c10-CLA induced excessive prostaglandin E2, adrenaline, corticosterone, glucagon and inflammatory factors in a dose-dependent manner, resulting in less heat release and oxygen consumption in Pai mice. Moreover, fibroblast growth factor 21 overproduction only in monoallelic Pai/wt mice indicates that it was sensitive to low doses of t10c12-CLA. These results suggest that chronic t10c12-CLA has system-wide effects on female health via synergistic actions of various hormones., (© 2024. The Author(s).)

Sex differences in opioid dependence (OD) are genetically influenced. We conducted genomewide gene-by-sex interaction scans for the DSM-IV diagnosis of OD in 8,387 African-American (AA) or European-American subjects (43.6% women; 4,715 OD subjects). Among AAs, 9 SNPs were genome-wide significant at ADGRV1 (adhesion G-protein-coupled receptor V1, lead-SNP rs2366929*(C/T), p = 1.5 × 10 -9 ) for sex-different risk of OD, with the rs2366929*C-allele increasing OD risk only for men. The top co-expressions in brain were between ADGRV1 and GRIK2 in substantia nigra and medullary inferior olivary nucleus, and between ADGRV1 and EFHC2 in frontal cortex and putamen. Significant sex-differential ADGRV1 expression from GTEx was detected in breast (Bonferroni-corrected-p < 0.002) and in heart (p < 0.0125), with nominal significance identified in brain, thyroid, lung, and stomach (p < 0.05). ADGRV1 co-expression and disease-enrichment analysis identifying the top 10 diseases showed strikingly sexually dimorphic risks. The enrichment and transcriptome analyses provided convergent support that ADGRV1 exerts a sex-different effect on OD risk. This is the first study to identify genetic variants contributing to sex differences in OD. It shows that ADGRV1 contributes to OD risk only in AA men, a finding that warrants further study.

Through unbiased transcriptomics and multiple molecular tools, transient downregulation of the Orthodenticle homeobox 2 (OTX2) gene was recently causatively associated with the development of depressive-like behaviors in a mouse model of early life stress. The analyses presented in this manuscript test the translational applicability of these findings by examining peripheral markers of methylation of OTX2 and OTX2-regulated genes in relation to measures of depression and resting-state functional connectivity data collected as part of a larger study examining risk and resilience in maltreated children. The sample included 157 children between the ages of 8 and 15 years (χ = 11.4, SD = 1.9). DNA specimens were derived from saliva samples and processed using the Illumina 450 K beadchip. A subset of children (N = 47) with DNA specimens also had resting-state functional MRI data. After controlling for demographic factors, cell heterogeneity, and three principal components, maltreatment history and methylation in OTX2 significantly predicted depression in the children. In terms of the imaging data, increased OTX2 methylation was found to be associated with increased functional connectivity between the right vmPFC and bilateral regions of the medial frontal cortex and the cingulate, including the subcallosal gyrus, frontal pole, and paracingulate gyrus-key structures implicated in depression. Mouse models of early stress hold significant promise in helping to unravel the mechanisms by which child adversity confers risk for psychopathology, with data presented in this manuscript supporting a potential role for OTX2 and OTX2-related (e.g., WNT1, PAX6) genes in the pathophysiology of stress-related depressive disorders in children.

Monoclonal antibodies (mAbs) are used as targeted therapies against cancers. These mAbs kill cancer cells via various mechanisms of actions. In this study, human embryonic stem cells (hESCs) was used as the immunogen to generate a panel of antibodies. From this panel of mAbs, A19 was found to bind both hESC and various cancer cell lines. The antigen target of A19 was identified as Erbb-2 and glycan analysis showed that A19 binds to a N-glycan epitope on the antigen. A19 was elucidated to internalize into cancer cells following binding to Erbb-2 and hence developed as an antibody-drug conjugate (ADC). Using ADC as the mechanism of action, A19 was able to kill cancer cells in vitro and delayed the onset of tumour formation in mice xenograft model. When compared to Herceptin, A19 binds to different isoforms of Erbb-2 and does not compete with Herceptin for the same epitope. Hence, A19 has the potential to be developed as an alternative targeted therapeutic agent for cancers expressing Erbb-2.

MRC-5 represents the most frequent human diploid cells (HDCs)-type cell substrate in the production of human viral vaccines. However, early-passage MRC-5 is diminishing and, due to both technical and ethical issues, it is extremely difficult to derive novel HDCs from fetal lung tissues, which are the common sources of HDCs. Our previous studies suggested that human umbilical cord may represent an alternative but convenient source of new HDCs. Here, we established a three-tiered cell banking system of a hUC-MSC line, designated previously as Cell Collection and Research Center-1 (CCRC-1). The full characterization indicated that the banked CCRC-1 cells were free from adventitious agents and remained non-tumorigenic. The CCRC-1 cells sustained its rapid proliferation even at passage 30 and were susceptible to the infection of a wide spectrum of viruses. Interestingly, the CCRC-1 cells showed much higher production of EV71 or Rubella viruses than MRC-5 and Vero cells when growing in serum-free medium. More importantly, the EV71 vaccine produced from CCRC-1 cells induced immunogenicity while eliciting no detectable toxicities in the tested mice. Collectively, these studies further supported that CCRC-1, and likely other hUC-MSCs as well, may serve as novel, safe and high-yielding HDCs for the production of human viral vaccines.

N-hydroxyphthalimide (NHPI), which is best known as an organocatalyst for efficient C-H activation, has been found to be oxidized by quinoid compounds to its corresponding catalytically active nitroxide-radical. Here, we found that NHPI can be isomerized into isatoic anhydride by an unusually facile two-step method using tetrachloro-1,4-benzoquinone (TCBQ, p-chloranil), accompanied by a two-step hydrolytic dechlorination of highly toxic TCBQ into the much less toxic dihydroxylation product, 2,5-dichloro-3,6-dihydroxy-1,4-benzoquinone (chloranilic acid). Interestingly, through the complementary application of oxygen-18 isotope-labeling, HPLC combined with electrospray ionization quadrupole time-of-flight and high resolution Fourier transform ion cyclotron resonance mass spectrometric studies, we determined that water was the source and origin of oxygen for isatoic anhydride. Based on these data, we proposed that nucleophilic attack with a subsequent water-assisted Lossen rearrangement coupled with rapid intramolecular addition and cyclization in two consecutive steps was responsible for this unusual structural isomerization of NHPI and concurrent hydroxylation/detoxication of TCBQ. This is the first report of an exceptionally facile double-isomerization of NHPI via an unprecedented water-assisted double-Lossen rearrangement under normal physiological conditions. Our findings may have broad implications for future research on hydroxamic acids and polyhalogenated quinoid carcinogens, two important classes of compounds of major chemical and biological interest.

Ripening is an important stage of fruit development. To screen the genes associated with pigment formation in tomato fruit, a suppression subtractive hybridization (SSH) cDNA library was constructed by using tomato fruit in the green ripe and break ripe stages, and 129 differential genes were obtained. Using redness as a screening marker, virus-induced gene silencing (VIGS) of the differential genes was performed with a sprout vacuum-infiltration system (SVI). The results showed that silencing the SlNAP7 gene affected the chloroplast development of tomato leaves, manifesting as a photo-bleaching phenotype, and silenced fruit significantly affected the accumulation of lycopene, manifested as a yellow phenotype. In our study, we found that silencing the SlNAP7 gene downregulates the expression of the POR and PORA genes and destroys the normal development of the chloroplast. The expression of related genes included in the lycopene biosynthesis pathway was not significantly changed, but lycopene accumulation was significantly reduced in tomato fruit. Perhaps it was caused by the destruction of the chromoplast, which leads to the oxidation of lycopene. The results show that the SlNAP7 gene influences chloroplast development and lycopene accumulation in tomato.

We found recently that intrinsic chemiluminescence (CL) could be produced by all 19 chlorophenolic persistent organic pollutants during environmentally-friendly advanced oxidation processes. Interestingly and unexpectedly, the strongest CL was produced not by the most-highly chlorinated pentachlorophenol (PCP), but rather by the less chlorinated 2,3,5,6-tetrachlorophenol (2,3,5,6-TeCP), one of the three tetrachlorophenol (TeCPs) isomers. However, it remains unclear what is the underlying molecular mechanism. Here we show that not only chlorinated quinoid intermediates, but more interestingly, semiquinone radicals were produced during the degradation of the three TeCPs and PCP by Fenton reagents, and the type and yield of which were found to be well correlated with CL generation. We propose that hydroxyl radical-dependent formation of more tetrachlorinated quinoids, quinone-dioxetanes and electronically excited carbonyl species might be responsible for the exceptionally strong CL production by 2,3,5,6-TeCP as compared to PCP and its two isomers. This is the first report showing the critical role of quinoid intermediates and semiquinone radicals in CL generation from polychlorinated phenols and Fenton system. These new findings may have broad chemical and environmental implications for future studies on remediation of other halogenated persistent aromatic pollutants by advanced oxidation processes.

Aim: To investigate the mechanisms underlying anticancer action of the benzimidazole acridine derivative N-{(1H-benzo[d]imidazol-2-yl)methyl}-2-butylacridin-9-amine(8m) against human colon cancer cells in vitro., Methods: Human colon cancer cell lines SW480 and HCT116 were incubated in the presence of 8m, and then the cell proliferation and apoptosis were measured. The expression of apoptotic/signaling genes and proteins was detected using RT-PCR and Western blotting. ROS generation and mitochondrial membrane depolarization were visualized with fluorescence microscopy., Results: 8m dose-dependently suppressed the proliferation of SW480 and HCT116 cells with IC50 values of 6.77 and 3.33 μmol/L, respectively. 8m induced apoptosis of HCT116 cells, accompanied by down-regulation of Bcl-2, up-regulation of death receptor-5 (DR5), truncation of Bid, cleavage of PARP, and activation of caspases (including caspase-8 and caspase-9 as well as the downstream caspases-3 and caspase-7). Moreover, 8m selectively activated JNK and p38 without affecting ERK in HCT116 cells. Knockout of JNK1, but not p38, attenuated 8m-induced apoptosis. In addition, 8m induced ROS production and mitochondrial membrane depolarization in HCT116 cells. Pretreatment with the antioxidants N-acetyl cysteine or glutathione attenuated 8m-induced apoptosis and JNK activation in HCT116 cells., Conclusion: The new benzimidazole acridine derivative, 8m exerts anticancer activity against human colon cancer cells in vitro by inducing both intrinsic and extrinsic apoptosis pathways via the ROS-JNK1 pathway.

Polyhalogenated quinones are a class of carcinogenic intermediates. We found recently that the highly reactive and biologically/environmentally important ·OH can be produced by polyhalogenated quinones and H₂O₂ independent of transition metal ions. However, it is not clear whether this unusual metal-independent ·OH producing system can induce potent oxidative DNA damage. Here we show that TCBQ and H₂O₂ can induce oxidative damage to both dG and dsDNA; but surprisingly, it was more efficient to induce oxidative damage in dsDNA than in dG. We found that this is probably due to its strong intercalating ability to dsDNA through competitive intercalation assays. The intercalation of TCBQ in dsDNA may lead to ·OH generation more adjacent to DNA. This is the first report that polyhalogenated quinoid carcinogens and H₂O₂ can induce potent DNA damage via a metal-independent and intercalation-enhanced oxidation mechanism, which may partly explain their potential genotoxicity, mutagenesis, and carcinogenicity.

Cocaine dependence (CD) and major depressive episode (MDE) frequently co-occur with poorer treatment outcome and higher relapse risk. Shared genetic risk was affirmed; to date, there have been no reports of genomewide linkage scans (GWLSs) surveying the susceptibility regions for comorbid CD and MDE (CD-MDE). We aimed to identify chromosomal regions and candidate genes susceptible to CD, MDE, and CD-MDE in African Americans (AAs) and European Americans (EAs). A total of 1896 individuals were recruited from 384 AA and 355 EA families, each with at least a sibling-pair with CD and/or opioid dependence. Array-based genotyping of about 6000 single-nucleotide polymorphisms was completed for all individuals. Parametric and non-parametric genomewide linkage analyses were performed. We found a genomewide-significant linkage peak on chromosome 7 at 183.4 cM for non-parametric analysis of CD-MDE in AAs (lod=3.8, genomewide empirical p=0.016; point-wise p=0.00001). A nearly genomewide significant linkage was identified for CD-MDE in EAs on chromosome 5 at 14.3 cM (logarithm of odds (lod)=2.95, genomewide empirical p=0.055; point-wise p=0.00012). Parametric analysis corroborated the findings in these two regions and improved the support for the peak on chromosome 5 so that it reached genomewide significance (heterogeneity lod=3.28, genomewide empirical p=0.046; point-wise p=0.00053). This is the first GWLS for CD-MDE. The genomewide significant linkage regions on chromosomes 5 and 7 harbor four particularly promising candidate genes: SRD5A1, UBE3C, PTPRN2, and VIPR2. Replication of the linkage findings in other populations is warranted, as is a focused analysis of the genes located in the linkage regions implicated here.

We recently reported that, in a European-American (EA) sample, the interaction between two cannabinoid receptor 1 gene (CNR1) variants significantly increased risk for drug dependence (DD), including cocaine dependence (CD). This study aimed to investigate directly the association between CNR1 and CD in four independent samples. Eight markers across the 45 kb CNR1 region and four large samples, ie, family-based European-American (EA) sample (n=734), case-control EA sample (n=862), family-based African-American (AA) sample (n=834) and case-control AA sample (n=619) were examined in the present study. We investigated the association of these markers with CD and cocaine-induced paranoia (CIP) in the EA family sample first, and then replicated positive results in the other three samples. The interaction between two independent CNR1 variants, ie, the G allele-containing genotypes of rs6454674 (SNP3(G+)), and the T/T genotype of rs806368 (SNP8(T)/T), significantly increased risk for CD in the EA family (P(GEE)=0.015) and EA case-control (P(regression)=0.003) samples. EA subjects with SNP3(G+) and SNP8(T)/T had higher risk to develop CD than those EA subjects with the other genotypes for these two SNPs (LR+ =1.4). The SNP3(G)-SNP8(T)haplotype also showed significant association (P=0.018) with CD in the EA case-control sample. SNP8-containing haplotypes showed significant association with both CD (P(global)=0.007) and CIP (P(global)=0.003) in the EA family sample. In the AA family sample, SNP8(T)/T significantly conferred higher risk for CD (P=0.019). We conclude that two independent CNR1 variants have significant interaction effects on risk for CD in EAs; they may also have effects on risk for CD in AAs.

Identification of the genetic factors that underlie stimulant responsiveness in animal models has significant implications for better understanding and treating stimulant addiction in humans. F(2) progeny derived from parental rat strains F344/NHsd and LEW/NHsd, which differ in responses to drugs of abuse, were used in quantitative trait locus (QTL) analyses to identify genomic regions associated with amphetamine-induced locomotion (AIL) and G-protein levels in the nucleus accumbens (NAc). The most robust QTLs were observed on chromosome 3 (maximal log ratio statistic score (LRS(max))=21.3) for AIL and on chromosome 2 (LRS(max)=22.0) for Galpha(i3). A 'suggestive' QTL (LRS(max)=12.5) was observed for AIL in a region of chromosome 2 that overlaps with the Galpha(i3) QTL. Novelty-induced locomotion (NIL) showed different QTL patterns from AIL, with the most robust QTL on chromosome 13 (LRS(max)=12.2). Specific unique and overlapping genomic regions influence AIL, NIL, and inhibitory G-protein levels in the NAc. These findings suggest that common genetic mechanisms influence certain biochemical and behavioral aspects of stimulant responsiveness.

The alcohol dehydrogenase (ADH) family constitutes one of the key sets of enzymes responsible for the oxidation of alcohol. The ADH4 gene, an important member of this family, is a functional and positional candidate for alcohol dependence. The present study aimed to investigate the relationship between ADH4 gene variation and alcohol dependence and drug dependence in European-Americans (EAs) and African-Americans (AAs). Seven single nucleotide polymorphisms (SNPs) spanning the ADH4 gene were genotyped in 365 healthy controls (317 EAs and 48 AAs) and 561 subjects (400 EAs and 161 AAs) affected with alcohol dependence and/or drug dependence (436 with alcohol dependence; 356 with drug dependence). Hardy-Weinberg equilibrium (HWE) for the genotype frequency distributions of these markers was tested in all phenotype groups to evaluate association between ADH4 gene variation and phenotypes and to fine-map the disease risk locus. The allele, genotype, and haplotype frequency distributions of these markers were compared between cases and controls to confirm the associations. The genotype frequency distributions of ADH4 markers were in HWE in EA controls, but were in Hardy-Weinberg disequilibrium (HWD) (ie, deviation from HWE) in EA cases. Among all markers, SNP2 (rs1042363) at exon 9 or SNP6 (rs1800759) at the promoter showed the greatest degree of HWD, among patients with either alcohol dependence or drug dependence. Significant differences between EA cases and controls were seen for genotype (10(-6)

Environmental or occupational exposure to mineral dusts, mainly silica and asbestos, is associated with an increased incidence of lung inflammation, fibrosis, and/or cancer. To better understand the molecular events associated with these pulmonary diseases, we attempted to identify genes that are regulated by mineral dusts. Using a differential display reverse transcription polymerase chain reaction technique and mRNAs of alveolar macrophages from both normal individuals and coal miners, we identified a novel mineral dust-induced gene named mdig, which had not been fully characterized. The expression of mdig mRNA was detected in alveolar macrophages from coal miners but not from normal subjects. The inducible expression of mdig could be observed in A549 cells exposed to silica particles in a time-dependent manner. The full-length mdig mRNA was expressed in human lung cancer tissues but was barely detectable in the adjacent normal tissues. In addition, a number of lung cancer cell lines constitutively express mdig. Alternative spliced transcripts of mdig were detected in some lung cancer cell lines. Silencing mdig mRNA expression in A549 lung cancer cells by siRNA-mediated RNA interference inhibits cell proliferation and sensitizes the cells to silica-induced cytotoxicity. These results suggest that the mdig gene may be involved in the regulation of cell growth and possibly the development of cancer.

Aim: To study the pharmacokinetics and accumulation of an Escherichia coli expressed His-tag fused recombinant human endostatin (rh-endostatin) in Rhesus monkeys., Methods: Rh-endostatin was iv or sc injected in Rhesus monkeys, and the rh-endostatin concentration in serum samples was determined by an enzyme immunoassay (EIA) method. The serum drug concentration-time data were analyzed by compartmental analysis using the practical pharmacokinetic program 3p97., Results: Following iv administration at a dose rate of 1.5, 4.5, and 13.5 mg/kg in rhesus monkeys, the concentration-time curves of rh-endostatin were best fitted to a three-compartment open model. AUC(0-infinity) linearly increased with dose, while Cls exhibited no significant difference among different dose groups. The terminal half-lives (lambda3) were 8+/-8, 3.1+/-1.4, and 20+/-14 h, respectively. After sc administration at a dose rate of 1.5 mg/kg, the concentration-time curve was best fitted to a two-compartment open model, with a terminal half-life (T(1/2beta)) of 8+/-3 h. Bioavailability following sc injection was approximately 70%+/-3%. After consecutive iv injection of rh-endostatin at a dose rate of 1.5 mg.kg(-1).d(-1) for 7 d, the AUC(0-24 h) substantially increased from 22+/-13 mg.h.L(-1) (d 1) to 50+/-29 mg.h.L(-1) (d 7), with an accumulation factor of 2.3+/-0.6 (P < 0.05)., Conclusion: The pharmacokinetic behavior of rh-endostatin in Rhesus monkeys complies with linear kinetics within the examined dose range. It tends to be accumulated in bodies after repeated administration at a dose level of 1.5 mg.kg(-1).d(-1) for more than 7 consecutive days.

The deleted in liver cancer (DLC-1) gene at chromosome 8p21-22 is altered mainly by genomic deletion or aberrant promoter methylation in a large number of human cancers such as breast, liver, colon and prostate and is known to have an inhibitory effect on breast and liver tumor cell growth. Given the high frequency of deletion involving region 8p21-22 in human non-small cell lung carcinoma (NSCLC), we examined alterations of DLC-1 in a series of primary tumors and tumor cell lines and tested effects of DLC-1 on tumor cell growth. A significant decrease or absence of the DLC-1 mRNA expression was found in 95% of primary NSCLC (20/21) and 58% of NSCLC cell lines (11/19). Transcriptional silencing of DLC-1 was primarily associated with aberrant DNA methylation, rather than genomic deletion as 5-aza-2'-deoxycytidine induced reactivation of DLC-1 expression in 82% (9/11) NSCLC cell lines showing downregulated DLC-1. It was further evidenced by an aberrant DLC-1 promoter methylation pattern, which was detected by Southern blotting in 73% (8/11) of NSCLC cell lines with downregulation of the gene. The transfer of DLC-1 into three DLC-1 negative cell lines caused a significant inhibition in cell proliferation and/or a decrease in colony formation. Furthermore, stable transfer of DLC-1 abolished tumorigenicity in nude mice of two cell lines, suggesting that DLC-1 plays a role in NSCLC by acting as a bona fide new tumor suppressor gene.

Aim: To optimize the antisense drug design by the combined method of phylogenetic analysis and secondary structure prediction and to get ideal candidates., Methods: The phylogenetic analysis and the secondary structure simulation were performed by computer. Oligodeoxynucleotides (ODN) were designed against the full-conserved blocks with low local reaction free energy of protein kinase C (PKC)-alpha mRNA. The in vitro effects of ODN were evaluated by human A549 lung carcinoma cells and mouse B16-BL6 melanoma cells, the expression of target mRNA was detected by in situ hybridization and RT-PCR. The in vivo effects of ODN were also evaluated by models of A549 xenografts in nude mice and B16 melanoma in mice., Results: Three ODN had significantly lower IC50 values than that of ISIS3521, the positive control, on A549 cells in vitro. Five ODN inhibited the growth of B16-BL6 cells with IC50 <100 nmol/L, while IC50 of ISIS3521 was >200 nmol/L. In situ hybridization and RT-PCR showed that the best candidate AP1261 inhibited the expression of PKC-alpha at mRNA level in a dose-dependent manner. AP1261 inhibited the growth of A549 and B16 tumors in vivo at 0.005-0.5 mg.kg(-1).d(-1). The inhibitory rate of AP1261 on A549 tumors was greater than that of ISIS3521 at the same dose. ISIS3521 did not affect the growth of B16 tumors., Conclusion: AP1261 may be of value as an antitumor agent or adjuvant and the combined method of phylogenetic analysis and secondary structure prediction is a potential helpful tool for antisense drug design.

The human DLC-1 (deleted in liver cancer 1) gene was cloned from a primary human hepatocellular carcinoma (HCC) and mapped to the chromosome 8p21-22 region frequently deleted in common human cancers and suspected to harbor tumor suppressor genes. DLC-1 was found to be deleted or downregulated in a significant number of HCCs. We expanded our investigations to other cancers with recurrent deletions of 8p22, and in this study examined alterations of DLC-1 in primary human breast tumors, human breast, colon, and prostate tumor cell lines. Genomic deletion of DLC-1 was observed in 40% of primary breast tumors, whereas reduced or undetectable levels of DLC-1 mRNA were seen in 70% of breast, 70% of colon, and 50% of prostate tumor cell lines To see whether DLC-1 expression affects cell growth and tumorigenicity, two breast carcinoma cell lines lacking the expression of endogenous gene were transfected with the DLC-1 cDNA. In both cell lines, DLC-1 transfection caused significant growth inhibition and reduction of colony formation. Furthermore, introduction of the DLC-1 cDNA abolished the in vivo tumorigenicity in nude mice, suggesting that the DLC-1 gene plays a role in breast cancer by acting as a bona fide tumor suppressor gene.

Aim: To study the pharmacokinetics (PK) and changes of kaolin partial thromboplastin time (KPTT) following single or multiple (7 d) dosing of a novel recombinant hirudin variant-2 (rHV-2) via the route of iv bolus injection (50 % of the total dose) plus infusion (the remained 50 % of the dose) in rhesus monkeys., Methods: A crossover design was applied to research the PK and KPTT profiles of rHV-2 after single (with total dose at 1, 3, and 6 mg/kg, respectively) and multiple dosing (3 mg/kg). An enzyme-linked immunosorbent assay (ELISA) method was utilized to determine the level of rHV-2 in plasma., Results: The concentration profiles of rHV-2 during or after administration were dependent both on the loading dose and the infusion rate. Mean Cmax after bolus in three single dose groups were 2.90, 9.78, and 15.68 mg/L, respectively. Infusions at rate of 8.35, 25, and 50 g/kg/h in 1 h resulted in steady-state levels of 0.73-0.86, 1.94-2.04, and 5.41-5.59 mg/L, respectively. The plasma rHV-2 levels during or after administration among doses were significantly different at most of the time points. Area under concentration-time curve (AUC) increased linearly with dose but systemic clearances were similar among different groups. KPTT was significantly prolonged (compared with baseline) at all dose levels, and trended to increase with dose., Conclusion: Both the loading dose and the infusion rate are very important for controlling the rHV-2 level, and the data may be helpful for optimizing dosage-regimen in clinical trials.

Aim: To optimize the design of antisense drug targeting protein kinase C-alpha (PKC-alpha) mRNA and obtain better antisense drugs than ISIS3521 that is undergoing clinical trials., Methods: RNAstructure (version 3.21, 1999) was utilized to predict the optimal and suboptimal secondary structures of human PKC alpha mRNA (GenBank, X52479), and 29 antisense phosphorothioate oligodeoxynucleotides (S-ODN) targeting the secondary structural elements, 3 partly matched S-ODN and 1 scrambled 3521 were designed. ISIS3521 was set as positive control. Mean (n = 3-5) 50% inhibitory effects on proliferation of A549 cells (IC50) of S-ODN were evaluated. Free energies (delta G degree 37) relating to the target secondary structural elements were calculated according to the nearest neighbor model. The quantitative structure-activity relationship (QSAR) analysis through multiple regression was obtained by SPSS., Results: Three S-ODN; (5'-AGCCCA-GCCGCTTGGCTGGG-3', 5'-AGGAGTGCAGCTGC-GTCAAG-3', 5'-TCAGAGGG-ACTGATGACTTT-3') had lower IC50[(48 +/- 7), (50 +/- 4), (64 +/- 2.7) nmol.L-1, respectively] than that of ISIS3521 [(81 +/- 25) nmol.L-1]. The number of bases comprising the target secondary structural element bulge loop, internal loop, and knot, the free energy of S-ODN (delta G degree 37S), and reaction (delta G degree 37R) were important parameters in QSAR equation. In the multiple regression, R was 0.68, P = 0.0193. Not tally with the equation, two S-ODN (5'-TCAAATGGAGG-CTGCCCGGC-3', 5'-AAAACGTCAGCCATGGTCCC-3') with favorable target structures and delta G degree 37 did not behave good activities., Conclusion: Computer aided design was helpful to obtain S-ODN with better in vitro effect than current positive drug. The degree of instability of secondary structural elements and delta G degree 37 were important factors for drug activity. Other important factors needed for further investigation.