[Association of urinary sodium excretion with fluid overload in patients with chronic kidney disease].

Objective: To investigate the relationship between urinary sodium excretion and fluid overload (FO) in non-dialysis patients with chronic kidney disease (CKD). Methods: Patients with CKD stage 1-4 who underwent bioelectrical impedance (BIA) in the Department of Nephrology, Jiangsu Province Hospital from December 2019 to January 2021 were recruited. All enrolled patients were categorized into two groups according to whether or not they develop FO. Further, clinical parameters were compared between the two groups. Spearman correlation analysis was used to investigate the association between over hydration/extracellular water (OH/ECW) and clinical characteristics. Multivariate logistic regression analysis was performed to evaluate the relationship between urinary sodium excretion and FO (FO was defined as OH/ECW≥7%). Results: A total of 385 patients with CKD stage 1-4 were finally included in the study, with a mean age of (46±15) years. There were 216 male cases (56.1%), and 150 cases (39.0%) existed FO. Spearman correlation analysis indicated that OH/ECW positively correlated with urinary sodium excretion (r=0.147, P=0.004), urinary protein excretion (r=0.555, P<0.001) and systolic blood pressure (r=0.241, P<0.001), but inversely related to estimated glomerular filtration rate (eGFR) (r=-0.111, P=0.030) and serum albumin (r=-0.659, P<0.001). After adjusting for confounding factors including age, systolic blood pressure, diabetes, urinary protein excretion, serum albumin, serum sodium, serum chlorine, urinary calcium excretion, urinary phosphorus excretion and use of diuretics, multivariate logistic regression analysis demonstrated that higher level of urinary sodium excretion was associated with increased risk of FO in patients with CKD (OR=1.005, 95%CI: 1.000-1.011, P=0.048). Conclusion: High urinary sodium excretion is independently associated with fluid FO in non-dialysis patients with CKD.

Barriers to early diagnosis and treatment of severely immunosuppressed patients with HIV-1 infection: A quantitative and qualitative study.

OBJECTIVES: To explore the barriers to early diagnosis of HIV infection and timely initiation of antiretroviral therapy (ART). METHODS: We assessed the annual number and proportion of ART-naïve people living with HIV infection (PLWH) with severe immunosuppression in Shenzhen, China, from 2008 to 2019. Selected ART-naïve PLWHs with severe immunosuppression who were seeking treatment for the first time in the hospital in 2019 were subjected to an in-depth interview. RESULTS: The proportion of severely immunosuppressed, ART-naïve PLWH decreased from 36.73% in 2008 to 8.94% in 2015, and then plateaued at approximately 10% from 2015 to 2019. Overall, 55 patients, 70% of whom were men who had sex with men, participated in the qualitative interviews. Ten of them delayed treatment after diagnosis, with a median [interquartile range (IQR)] interval of 5.83 (3.98-8.54) years between diagnosis and ART. More than 80% of the patients reported casual sexual contact within a median period of 6 years and with a median (IQR) of nine (4-20) casual sex partners. The major barriers to HIV testing and diagnosis included lack of knowledge about HIV and high-risk behaviours, low awareness about HIV testing, and resistance to HIV testing. The major barriers to ART initiation included lack of knowledge about the importance of ART and change of national ART eligibility policy, and HIV-related stress. CONCLUSIONS: The number of PLWHs with severe immunosuppression who seek treatment remains high in Shenzhen, China. Thus, current HIV-related care programmes targeting access to early diagnosis and treatment need to be improved.

miR-142-3p Inhibits the Metastasis of Hepatocellular Carcinoma Cells by Regulating HMGB1 Gene Expression.

BACKGROUND: Non-coding small RNAs are involved in organism development, and their aberrant regulation induces various diseases, including hepatocellular carcinoma (HCC), but their exact mechanisms have not been determined. OBJECTIVE: The aim was to investigate the role of miR-142-3p on HMGB1 expression in hepatocellular carcinoma. METHODS: Expression levels of miR-142-3p in HCC tissues and cultured cells were measured by RT-PCR. The invasion and metastasis abilities of HepG2 cells according to Transwell migration and invasion assays, and protein expression was measured by western blotting. RESULTS: The present study reported that miR-142-3p promotes the invasion and migration of HCC cells. miR-142-3p levels are lower in HCC tissues than in adjacent non-cancerous tissues, suggesting a tumor suppressor role for miR-142-3p. Highmobility group box protein 1 (HMGB1) is an oncogene that promotes the metastasis of HCC. miR-142-3p or HMGB1 knockdown alone inhibits the invasion and migration of HCC cells, and HMGB1 overexpression impedes the effect of miR-142-3p. Further studies showed that HMGB1 is a direct target gene of miR-142-3p in HCC. miR-142-3p represses HMGB1 gene transcription by directly binding to the 3' untranslated region (UTR) of HMGB1, thereby inhibiting cancer cell invasion and migration. CONCLUSION: This study, for the first time, reports that miR-142-3p is a novel tumor suppressor that inhibits the invasion and migration of HCC cells by directly regulating gene transcription of HMGB1. Thus, miR-142-3p may be a potential diagnostic and therapeutic biomarker for HCC patients.

Quantitative magnetic resonance imaging analysis correlates with surgical outcome of cervical spondylotic myelopathy.

STUDY DESIGN: Prospective study. OBJECTIVES: To investigate whether preoperative and postoperative changes of signal intensity (SI) and transverse area (TA) of the spinal cord reflect the surgical outcome in patients with cervical spondylotic myelopathy (CSM). SETTING: The Second Hospital of Tangshan, Tangshan, Hebei, China. METHODS: In 45 consecutive prospective patients, magnetic resonance imaging (MRI) was performed preoperatively and 3 months postoperatively. The Japanese Orthopedic Association (JOA) scale was used to quantify the neurological status at admission and of at least 12-month follow-up. Preoperative and postoperative TA of the spinal cord at the site of maximal compression and grayscale of signal intensity (GSI) were measured using the image analysis software. Ratio of transverse area (RTA) and ratio of grayscale of signal intensity (RGSI) were used to assess the extent of spinal cord re-expansion and extent of SI regression. Preoperative status and postoperative recovery were assessed in relation to MRI parameters preoperatively and postoperatively using univariate and multivariate analysis. RESULTS: Higher baseline JOA scores were associated with larger TA. Greater recovery rate was associated with larger preoperative and postoperative TA, along with greater RTA. Recovery rate negatively correlated with RGSI and age. Higher baseline JOA score was associated with greater recovery rate. RGSI negatively correlated with RTA. Multivariate stepwise regression analysis showed that the optimal combination of surgical outcome predictors included age, postoperative TA and RGSI. CONCLUSION: Quantitative MRI analysis in CSM may provide reliable information for the prediction of the postoperative outcome of CSM patients. MRI indicators of good outcome include the larger postoperative TA and greater RGSI.

Quantitative magnetic resonance imaging analysis correlates with surgical outcome of cervical spinal cord injury without radiologic evidence of trauma.

STUDY DESIGN: Prospective study. OBJECTIVES: To investigate whether pre- and post-operative changes of signal intensity (SI) and transverse area (TA) of the spinal cord on T2-weighted magnetic resonance imaging (MRI) reflect the surgical outcome in patients with spinal cord injury (SCI) without radiologic evidence of trauma (SCIWORET). SETTING: The Third Hospital of Hebei Medical University, Shijiazhuang, Hebei, China. METHODS: In 36 consecutive prospective patients, MRI was performed pre-operatively and 3 months post-operatively. The Japanese Orthopaedic Association (JOA) scale and the American Spinal Cord Injury Association (ASIA) motor score (AMS) were used to quantify neurologic status at admission and at least 12-month follow-up. Pre- and post-operative TA, range of signal intensity (RSI), grayscale of signal intensity (GSI) and prevertebral hyperintensities (PVHs) were measured using the image analysis software. Pre-operative status and post-operative recovery were assessed in relation to MRI parameters pre- and post-operatively using univariate and multivariate analysis. RESULTS: Pre-operative JOA and AMS score negatively correlates RSI, GSI and PVH. There was no significant correlation between pre-operative TA and pre-operative JOA and AMS. Recovery rate with JOA negatively correlates pre-operative RSI, post-operative RSI, pre-operative GSI, post-operative GSI and PVH. There was a significant negative correlation between recovery rate with AMS and pre-operative RSI, post-operative GSI and PVH. From these results of multivariate stepwise regression analysis, the predictors of surgical outcomes are pre-operative GSI and pre-operative RSI. CONCLUSION: Quantitative MRI analysis may provide reliable information for the prediction of the initial neurological status and surgical outcome of patients with SCIWORET.

Targeting Epstein-Barr virus oncoprotein LMP1-mediated glycolysis sensitizes nasopharyngeal carcinoma to radiation therapy.

Our goal in this work was to illustrate the Epstein-Barr virus (EBV)-modulated global biochemical profile and provide a novel metabolism-related target to improve the therapeutic regimen of nasopharyngeal carcinoma (NPC). We used a metabolomics approach to investigate EBV-modulated metabolic changes, and found that the exogenous overexpression of the EBV-encoded latent membrane protein 1 (LMP1) significantly increased glycolysis. The deregulation of several glycolytic genes, including hexokinase 2 (HK2), was determined to be responsible for the reprogramming of LMP1-mediated glucose metabolism in NPC cells. The upregulation of HK2 elevated aerobic glycolysis and facilitated proliferation by blocking apoptosis. More importantly, HK2 was positively correlated with LMP1 in NPC biopsies, and high HK2 levels were significantly associated with poor overall survival of NPC patients following radiation therapy. Knockdown of HK2 effectively enhanced the sensitivity of LMP1-overexpressing NPC cells to irradiation. Finally, c-Myc was demonstrated to be required for LMP1-induced upregulation of HK2. The LMP1-mediated attenuation of the PI3-K/Akt-GSK3beta-FBW7 signaling axis resulted in the stabilization of c-Myc. These findings indicate a close relationship between EBV and glycolysis in NPC. Notably, LMP1 is the key regulator of the reprogramming of EBV-mediated glycolysis in NPC cells. Given the importance of EBV-mediated deregulation of glycolysis, anti-glycolytic therapy might represent a worthwhile avenue of exploration in the treatment of EBV-related cancers.

Vascularized dorsal digital fascial flap improves flexor tendon repairs.

We report a new method of flexor tendon repair in zone II using a standard modified Kessler technique combined with a vascularized dorsal fascial flap from the finger pedicled on a dorsal cutaneous branch of the proper digital artery, which is placed as a mechanical barrier between the flexor digitorum superficialis and profundus tendons. The functional outcomes of 14 patients (Group A) with flexor tendon repairs in zone II by this new technique were compared with those of 32 patients (Group B) with flexor tendon repairs in zone II using a standard modified Kessler technique only. Patients in Group A had a higher proportion of excellent results (on the modified Strickland system) and more movement in the distal interphalangeal joint than the patients in Group B.

Antiviral role of toll-like receptor-3 agonists against seasonal and avian influenza viruses.

The divergence and antigenic shifts in influenza viruses represent significant challenges for the development of effective vaccines and antiviral drugs against influenza viruses. In view of current challenges and/or deficiencies in the influenza pandemic influenza preparedness, novel antiviral strategies which are robust and can respond to constant viral mutations, are particularly needed to combat future pandemic threats. Toll-like receptor-3 (TLR-3) is an integral part of the host's innate immune system and serves as an important signaling pathway for the recognition of dsRNA for the triggering of antiviral and inflammatory responses to combat viral infections. This review examines dsRNA including Poly ICLC and liposome-encapsulated Poly ICLC (LE Poly ICLC) as TLR-3 agonists for their antiviral activity against seasonal and highly pathogenic avian influenza (HPAI) viruses. Furthermore, their roles in attenuating the antiviral and inflammatory cytokines in the host will also be explored. Preclinical studies in experimental animals suggest Poly ICLC and liposome-encapsulated Poly ICLC are safe and offer broad-spectrum protection against both seasonal and HPAI viruses, as well as other respiratory viruses including respiratory syncytial virus and SARS. Preliminary results from recent studies suggest these drugs up-regulate the production of interferons (-alpha, -beta, and -gamma), and tumor necrosis factor (TNF-alpha) but downregulate some proinflammatory cytokines including IL-2 and IL-4. Taken together, these results suggest these TLR-3 agonists have a promising role to play as safe, effective and broad-spectrum anti-influenza drugs that could complement other antiviral drugs to combat seasonal, zoonotic and pandemic influenza viruses. The clinical safety of these drugs and their efficacy in pre-clinical studies may provide sufficient justification for regulatory agencies to consider their fast track development for use in future outbreaks of pandemic influenza or of other emerging respiratory pathogens.

Activation of toll-like receptor signaling pathway for protection against influenza virus infection.

This study aims to evaluate the antiviral role of nucleic acid-based agonists for the activation of toll-like receptor (TLR) signaling pathways, and its protective role in respiratory influenza A virus infections. TLR-3 is expressed on myeloid dendritic cells, respiratory epithelium, and macrophages, and appears to play a central role in mediating both the antiviral and inflammatory responses of the innate immunity in combating viral infections. Influenza viruses can effectively inhibit the host's ability to produce interferons, and thereby suppress the immune system's antiviral defence mechanisms. Poly ICLC is a synthetic double stranded RNA comprising of polyriboinosinic-poly ribocytidylic acid (Poly IC) stabilized with l-lysine (L) and carboxymethylcellulose (C). Poly ICLC and liposome-encapsulated Poly ICLC (LE Poly ICLC) are TLR-3 agonists and are potent inducer of interferons and natural killer cells. Intranasal pre-treatment of mice with Poly ICLC and LE Poly ICLC provided high level of protection against lethal challenge with a highly lethal avian H5N1 influenza (HPAI) strain (A/H5N1/chicken/Henan clade 2), and against lethal seasonal influenza A/PR/8/34 [H1N1] and A/Aichi/2 [H3N2] virus strains. The duration of protective antiviral immunity to multiple lethal doses of influenza virus A/PR/8/34 virus had been previously found to persist for up to 3 weeks in mice for LE Poly ICLC and 2 weeks for Poly ICLC. Similarly, pre-treatment of mice with CpG oligonucleotides (TLR-9 agonist) was also found to provide complete protection against influenza A/PR/8/34 infection in mice. RT-PCR analysis of lung tissues of mice treated with Poly ICLC and LE Poly ICLC revealed upregulation of TLR-3 mRNAs gene expression. Taken together, these results do support the potential role of TLR-3 and TLR-9 agonists such as Poly ICLC and LE Poly ICLC in protection against lethal seasonal and HPAI virus infection.

Myostatin antisense RNA-mediated muscle growth in normal and cancer cachexia mice.

Myostatin is a negative regulator of myogenesis, and inactivation of myostatin leads to muscle growth. Here we have used modified RNA oligonucleotides targeting the myostatin mRNA and examined the therapeutic potential in normal and cancer cachexia mouse models. We found that the RNA oligonucleotides could suppress the myostatin expression in vivo, leading to the increase in muscle growth both in normal and cachectic mice. We also established that the effect of myostatin inhibition caused by the RNA oligonucleotides may be through the MyoD pathway, as evidenced by a significant upregulation of MyoD expression. Taken together, these results demonstrate the feasibility using antisense strategy for the treatment of muscle wasting conditions.

Effect of RNA oligonucleotide targeting Foxo-1 on muscle growth in normal and cancer cachexia mice.

Foxo-1, a member of the Foxo forkhead type transcription factors, is markedly upregulated in skeletal muscle in energy-deprived states such as fasting, cancer and severe diabetes. In this study, we target the Foxo-1 mRNA in a mouse skeletal myoblast cell line C2C12 and in vivo models of normal and cancer cachexia mice by a Foxo-1 specific RNA oligonucleotide. Our results demonstrate that the RNA oligonucleotide can reduce the expression of Foxo-1 in cells and in normal and cachectic mice, leading to an increase in skeletal muscle mass of the mice. In search for the possible downstream target genes of Foxo-1, we show that when Foxo-1 expression is blocked both in cells and in mice, the level of MyoD, a myogenic factor, is increased while a muscle negative regulator GDF-8 or myostatin is suppressed. Taken together, these results show that Foxo-1 pays a critical role in development of muscle atrophy, and suggest that Foxo-1 is a potential molecular target for treatment of muscle wasting conditions.

Nucleic acid-based antiviral drugs against seasonal and avian influenza viruses.

Influenza viruses are etiological agents of deadly flu that continue to pose global health threats, and have caused global pandemics that killed millions of people worldwide. The availability of neuraminidase inhibitors and attenuated vaccines improves our ability to defend against influenza, but their benefits can be significantly limited by drug-resistance and virus mutations. Nucleic acid-based drugs may represent a promising class of antiviral agents that could play a role in the prevention and treatment of influenza. Efficacy studies in animals have shown that ds RNA, such as poly ICLC can provide effective and broad-spectrum prophylaxis against lethal challenges against various strains of influenza A virus. Furthermore, similar level of antiviral protection in mice can be provided by using short fragments of oligonucleotides that induce antiviral immunity. Finally, influenza virus expression can also be specifically inhibited or suppressed using antisense oligonucleotides that bind to viral mRNA encoding key viral proteins. The versatility and potency of nucleic acid-based drugs make them potential drug candidates for used in seasonal or pandemic influenza situations.

Gene profiling after knocking-down expression of nucleostemin in Hela cells using oligonucleotide DNA microarray.

Nucleostemin (NS) is preferentially and exclusively expressed in the stem cells and cancer cells, but not in differentiated adult tissues and cells. NS is likely to take part in controlling the proliferation and differentiation switch in stem cells and progenitor cells. Its deregulation in cancer also contributes to the elevated proliferation and undifferentiation of cancer cells. However, the mechanisms by which NS helps to maintain both cancer and stem cells in undifferentiated state remain unclear. In this study, we carried out gene profilings using oligonucleotide DNA microarray after knocking down the expression of NS in Hela cells. Of the 21,329 genes, 200 genes were found differentially expressed in NS silenced Hela cells with > 2 fold ratio (either > 2 or < 0.5). Category analysis indicated these differential genes were mainly related with cancer pathogenesis, cell death, cell growth and proliferation. NS related gene pathway analysis suggested NS was mostly involved in the networks of cell cycle and differentiation controls. p53 may not be the only partner of NS in its regulated pathways. c-Myc may directly or indirectly interact with it to control the proliferation and differentiation switch in cancer cells. Our study provides a general view of the NS-target genes, and indicates the possible pathways in which NS plays its role in proliferation control.

Catalytic DNA: a novel tool for gene suppression.

RNA, as an intermediate in the production of every gene encoded protein and the genetic material of many pathogenic viruses, presents an attractive target for both biological and therapeutic manipulation. Despite its extensive involvement in living systems, its chemical diversity based on four units is relatively low compared with protein. This provides the opportunity for a generic approach to targeting with specificity based on primary structure rather than complex higher order structures. This form of recognition occurs naturally in complementary nucleic acids, due to an ability to bind their single stranded target through Watson-Crick interactions. The most established nucleic acid based approach to gene suppression at the RNA level is through antisense oligodeoxynucleotides (ODNs). These compounds form heteroduplex with target RNA which are thought to either block its function or mediate its destruction by activation of RNase H. Alternatively, RNA can be targeted by catalytic RNA such as the hammerhead ribozyme. Ribozymes have the advantage of being equipped with their own RNA cleavage apparatus and are therefore independent of host nuclear protein activity. At present, the utility of ribozyme oligonucleotides is restricted by the relative difficulty synthesising active molecules with sufficient resistance to nuclease degradation. Recently the power of in vitro selection has been used to evolve catalytic DNA sequences with RNA cleavage specificity and activity rivalling the very best ribozymes, while maintaining the more robust chemistry of an ODN. These deoxyribozymes or DNAzymes have tremendous potential as gene suppression agents for both target validation and therapeutic applications. A number of studies evaluating the biological activity of these compounds have shown promising results. However, as with other oligonucleotide based strategies, future exploitation of this approach may depend on accessory technology to assist with the accessibility of a target which is folded by its own secondary structure and hidden within the intracellular compartment.

Suppression of platelet-type 12-lipoxygenase activity in human erythroleukemia cells by an RNA-cleaving DNAzyme.

Human platelet-type 12-lipoxygenase (12-LOX) and its metabolites play a crucial role in tumor angiogenesis. A "10-23" deoxyribozyme (DNAzyme) and its phosphorothioate-modified version were designed and synthesized against the 12-LOX mRNA. Both DNAzymes were able to cleave their substrate efficiently in a time- and concentration-dependent manner in vitro. Under a multiple turnover condition, both performed well at 37 degrees C, showing the k(cat) of 1 and 0.26 min(-1), respectively. The phosphorothioate modification of the DNAzyme significantly increased its stability in cells without a substantial loss of kinetic efficiency in vitro. In a cell culture system, transfection of the DNAzymes into HEL cells resulted in a significant down-regulation of the 12-LOX mRNA. Furthermore, the cell extracts from the DNAzyme-transfected cells exhibited a marked reduction in the 12-LOX enzyme activity. The present results indicated the potential use of DNAzyme technology for gene function study and cancer therapy.

Fractionated irradiation combined with carbogen breathing and nicotinamide of two human glioblastomas grafted in nude mice.

This study addressed the potential radiosensitizing effect of nicotinamide and/or carbogen on human glioblastoma xenografts in nude mice. U-87MG and LN-Z308 tumors were irradiated with either 20 fractions over 12 days or 5 fractions over 5 days in air-breathing mice, mice injected with nicotinamide, mice breathing carbogen, or mice receiving nicotinamide plus carbogen. The responses to treatment were assessed using local control and moist desquamation. In U-87MG tumors, the enhancement ratios (ERs) at the radiation dose required to produce local tumor control in 50% of the treated mice (TCD(50)) with nicotinamide and/or carbogen ranged from 1.13 to 1.24 for irradiation in 20 fractions over 12 days. In LN-Z308 tumors, the ERs at the TCD(50) with nicotinamide and/or carbogen ranged from 1.22 to 1.40 for irradiation in 5 fractions over 5 days and from 1.11 to 1.30 in 20 fractions over 12 days, respectively. Skin injury was slightly enhanced, with ERs ranged from 1.06 to 1.15 when radiation was combined with carbogen and/or nicotinamide. Thus carbogen and nicotinamide can slightly improve the radiation response of human glioblastoma xenografts.

Induced p21WAF1 expression acts to reverse myc myelomonocytic cell transformation.

Two murine myelomonocytic cells lines were used to examine p21WAF1 expression in myc-induced cell transformation. tEMmyc4 and FDLV are two v-myc-transformed immortalised myeloid cell lines exhibiting different transformed phenotypes. FDLV cells were derived from the transduction of v-myc into FDC-P1 cells and retain growth factor (IL-3) dependence, whereas tEMmyc4 cells were derived from the transduction of embryonal monocytes with v-myc and are growth factor-independent, constitutively express endogenous CSF-1, and are highly tumorigenic in syngeneic mice. Both cell lines were found to exhibit low p21WAF1 expression. When examined in tEMmyc4 cells, neither the p53-dependent pathway (mitomycin C or exogenous p53) nor p53-independent pathway (TPA or growth factor, CSF-1, stimulation) acted to increase p21WAF1 levels. Growth factor (IL-3) withdrawal, shown to reduce p21WAF1 levels in parental FDC-P1 cells, failed to do this in FDLV cells. The dependence of p21WAF1 expression on v-myc was further demonstrated by showing that a v-myc-targeted ribozyme, which acts to decrease v-myc RNA, increased p21WAF1 levels in tEMmyc4 cells. Enforced expression of exogenous p21WAF1 in tEMmyc4 cells with dysfunctional growth cycle (including growth arrest and increased susceptibility to apoptosis) was examined. p21WAF1 partially restored cell cycle regulation and apoptosis as well as inhibited the delayed cell cycle progression and apoptosis induced by mitomycin C or serum withdrawal. These results show p21WAF1 expression to be affected by v-myc and a restoration of p21WAF1 expression to partially reverse myc-mediated transformation.

Unlabelled iododeoxyuridine increases the cytotoxicity and incorporation of [1251]-iododeoxyuridine in two human glioblastoma cell lines.

Iododeoxyuridine (IUdR), labelled with radioiodines emitting Auger, alpha or beta- radiation, has been proposed as a therapeutic tool in the treatment of cancer. However, the low per cent incorporation in tumour cells and limited cytotoxicity are major obstacles for such an application. Using unlabelled IUdR as a modulator, we have studied the in vitro cytotoxicity of [125I]-IUdR in two human glioblastoma cell lines. Surprisingly, an enhanced cytotoxicity of [125I]-IUdR was observed in the presence of 0.3-10 microM concentrations of unlabelled IUdR in U251 glioblastoma cells and to a lesser extent in LN229 cells. The presence of unlabelled IUdR unexpectedly increased the incorporation of [125I]-IUdR in both cell lines. Thymidine competitively blocked the cytotoxic effects of combined unlabelled and [125I]-labelled IUdR in these cells and DNA-incorporation of radiolabelled IUdR.

The influence of arm length asymmetry and base substitution on the activity of the 10-23 DNA enzyme.

A small oligodeoxyribonucleotide derived from in vitro selection has been shown to be capable of efficient sequence-specific cleavage of RNA at purine-pyrimidine junctions. As the reaction readily takes place under simulated physiologic conditions, this molecule described as the 10-23 general purpose RNA-cleaving DNA enzyme, has potential as a therapeutic agent. To further explore the character of this prototype, we examined the influence of base substitution and binding arm length asymmetry on its RNA cleaving activity. Surprisingly, substitution of the proximal nucleotide on the 3'-arm, to allow nonstandard Watson-Crick interactions, was found in some instances to improve the cleavage reaction rate. Although the identity of the unpaired purine in the RNA substrate cleavage site was found to have only a subtle influence on the rate of catalysis, with a slight decrease observed when a G at this position was changed to an A, nucleotide substitution (G to C) in the core motif at position 14 was found to completely abolish catalysis. The effect of arm length reduction varied with RNA substrate sequence and extent of helix asymmetry. Where the cleavage rate of one substrate was impaired by truncation of the deoxyribozymes 5'-arm (6 bp), the same modification in reactions with a different sequence produced a rate enhancement. Truncation of the 3'-arm, however, had no effect on the reaction rate of the one substrate tested yet nearly halved the cleavage rate in another substrate.

Catalytic nucleic acids: from lab to applications.

Since the discovery of self-cleavage and ligation activity of the group I intron, the expansion of research interest in catalytic nucleic acids has provided a valuable nonprotein resource for manipulating biomolecules. Although a multitude of reactions can be enhanced by this class of catalyst, including trans-splicing activity of the group I intron (which could be applied to gene correction), RNA-cleaving RNA enzymes or "ribozymes" hold center stage because of their tremendous potential for mediating gene inactivation. This application has been driven predominantly by the "hammerhead" and "hairpin" ribozymes as they induce specific RNA cleavage from a very small catalytic domain, allowing delivery either as a transgene expression product or directly as a synthetic oligonucleotide. Although advances in the development of RNA modifications have improved the biological half-life of synthetic ribozymes, their use is restricted by the mechanistic dependence on conserved 2'OH-moieties. Recently a new class of catalytic nucleic acid made entirely of DNA has emerged through in vitro selection. DNA enzymes or deoxyribozyme with extraordinary RNA cleavage activity has already demonstrated their capacity for gene suppression both in vitro and in vivo. These new molecules, although rivaling the activity and stability of synthetic ribozymes, are limited equally by inefficient delivery to the intracellular target RNA. The challenge of in vivo delivery is being addressed with the assessment of a variety of approaches in animal models with the aim of bringing these compounds closer to the clinic.