Nuclease resistant ribozymes with high catalytic activity.

Hammerhead ribozymes are efficient RNA enzymes characterized by a typical hammerhead secondary structure and a number of conserved bases. Little is known about the role of the ribose-phosphate backbone, although it is obviously important since a DNA molecule with the same base sequence is not a catalyst. Here we describe the synthesis of artificial ribozymes where modified (2'-O-allyl- and 2'-O-methyl-) ribonucleotides substitute for the corresponding ribonucleotides. A systematic analysis of partially substituted polymers identified a minimum set of six non-contiguous positions where insertion of modified ribonucleotides strongly affects catalytic activity. Surprisingly, ribozymes completely substituted except for these six ribonucleotides are still very active. These molecules efficiently cleave in trans target RNAs in a sequence-specific way, but, unlike RNA ribozymes, are very resistant to nuclease degradation and are very stable in serum. These properties make such synthetic polymers potentially useful for in vivo gene expression studies and therapeutic applications.     

Establishment of a doxycycline-regulated cell line with inducible,doubly-stable expression of the wild-type p53 gene from p53-deleted hepatocellular carcinoma cells

p53 is important in the development of hepatocellular carcinoma (HCC) and in therapeutic approaches, but the mechanism whereby it inhibits HCC growth is still unclear. The aim of the present study was to establish a HCC cell system in which p53 levels can be regulated. Full-length wild-type p53 cDNA obtained by PCR was cloned into a retroviral response vector controlled by the tetracycline responsive element (RevTRE-p53). The regulatory vectors RevTet-Off and RevTRE-p53 were transfected into a packaging cell line, PT67. Hep3B cells in which the p53 gene was deleted were infected with RevTet-Off viral particles from the PT67. Three G418-resistant cell clones with high luciferase expression and low background were infected with RevTRE-p53. By screening dozens of RevTRE-p53-infected clones with hygromycin we identified the one with the highest expression of p53 and the lowest background after doxycycline treatment. The results showed that p53 expression in this cell clone could be simply turned on or off by removing or adding doxycycline. Furthermore, it was found that the level of p53 protein was negatively and sensitively related to the doxycycline concentration. In conclusion, we have established a HCC cell line in which p53 expression can be switched on or off and regulated in a dose- and time-dependent manner.     

Detecting substitution-rate heterogeneity among regions of a nucleotide sequence

Likelihood-ratio statistics are proposed to test for heterogeneity in nucleotide substitution rate among regions of a DNA sequence. The tests examine three-sequence phylogenies, and two specific tests are proposed: a test to detect rate heterogeneity among genic regions within a sequence, over all evolutionary lineages; and a test to detect rate heterogeneity among regions in a specific evolutionary lineage. Simulations examine the ability of tests to detect a single region that varies in nucleotide substitution rate relative to the remainder of the sequence. A 50-bp region with a fivefold substitution-rate increase can be detected > or = 90% of the time when it is found in all three lineages of the phylogeny, and a 50-bp region of fivefold rate increase can be detected with approximately 70% power when it is found in only one evolutionary lineage. Simulation also examines the effect of transition- and transversion-rate differences. The tests are applied to published DNA sequences. While the tests are powerful, significant results can be difficult to interpret biologically.      

Sequential NMR assignments of labile protons in DNA using two-dimensional nuclear-Overhauser-enhancement spectroscopy with three jump-and-return pulse sequences

Two-dimensional nuclear Overhauser enhancement (NOESY) spectra of labile protons were recorded in H2O solutions of a protein and of a DNA duplex, using a modification of the standard NOESY experiment with all three 90 degree pulses replaced by jump-and-return sequences. For the protein as well as the DNA fragment the strategically important spectral regions could be recorded with good sensitivity and free of artifacts. Using this procedure, sequence-specific assignments were obtained for the imino protons, C2H of adenine, and C4NH2 of cytosine in a 23-base-pair DNA duplex which includes the 17-base-pair OR3 repressor binding site of bacteriophage lambda. Based on comparison with previously published results on the isolated OR3 binding site, these data were used for a study of chain termination effects on the chemical shifts of imino proton resonances of DNA duplexes.     

Direct genomic fluorescent on-line sequencing and analysis using in vitro amplification of DNA.

In vitro amplification of genomic DNA and total RNA, as well as recombinant DNA, using one fluorescently labelled and one unlabelled primer during amplification, together with on-line analysis of the products on the EMBL fluorescent DNA sequencer, is described. Further is reported direct sequencing of fluorescently labelled amplified probes by solid-phase chemical degradation, without subcloning and purification steps involved. At present up to 350 bases in 4 hours are determined with this technique. The fluorescent dye and its bond to the oligonucleotide are stable during the amplification cycles, and do not interfere with the enzymatic polymerization. High sensitivity of the detection device, down to 10(-18) moles, corresponding to less than 10(6) molecules makes possible analyses of the non-radioactive amplified probes after only 10 amplification cycles, starting with about 5 x 10(4) copies of recombinant DNA.     

Predictors of mortality of patients newly diagnosed with clinical type 2 diabetes: a 5-year follow up study

Background

At diabetes diagnosis major decisions about life-style changes and treatments are made based on characteristics measured shortly after diagnosis. The predictive value for mortality of these early characteristics is widely unknown. We examined the predictive value of patient characteristics measured shortly after diabetes diagnosis for 5-year all-cause and cardiovascular mortality with special reference to self-rated general health.

Methods

Data were from a population-based sample of 1,323 persons newly diagnosed with clinical diabetes and aged 40 years or over. Possible predictors of mortality were investigated in Cox regression models.

Results

Multivariately patients who rated their health less than excellent experienced increased all-cause and cardiovascular mortality. These end-points also increased with sedentary life-style, relatively young age at diagnosis and presence of cardiovascular disease (CVD) at diagnosis. Further predictors of all-cause mortality were male sex, low body mass index and cancer, while cardiovascular mortality increased with urinary albumin concentration.

Conclusions

We found that patients who rated their health as less than excellent had increased 5-year mortality, similar to that of patients with prevalent CVD, even when biochemical, clinical and life-style variables were controlled for. This finding could motivate doctors to discuss perceptions of health with newly diagnosed diabetic patients and be attentive to patients with suboptimal health ratings. Our findings also confirm that life-style changes and optimizing treatment are particularly relevant for relatively young and inactive patients and those who already have CVD or (micro)albuminuria at the time of diabetes diagnosis.     

Durable remission of diabetes after bariatric surgery: What is the underlying pathway?

Background: In the past, type 2 diabetes mellitus (DM) was regarded as a progressive, incurable disease for which palliative therapy could not, over the long term, prevent the associated amputations, blindness, renal failure, and early mortality. This is no longer true. Full and durable remission of type 2 DM, with major decreases in morbidity and mortality, is now achieved regularly with several types of surgery that reduce contact between food and the foregut.Objectives: The aims of this article are to review the impact of bariatric surgery on obesity, remission of DM, and obesity-related morbidity and mortality, and the possible mechanisms for this advance.Methods: This article is based on our 2 meta-analyses of the literature published through April 30, 2006, as well as the most significant reports in the bariatric surgical literature that have been published in English since April 30, 2006. The studies included in our second meta-analysis provided the details of the methodology for the present literature review, including the levels of evidence.Results: Results of our 2 meta-analyses were published previously. Briefly, the analyses revealed that the clinical and laboratory manifestations of type 2 DM resolved or improved in most of the patients who underwent bariatric surgery; the responses were greatest in the patients who lost the most excess body weight; and the improvements were maintained for ≥2 years. The studies reported that intestinal operations such as gastric bypass reduced contact between food and the foregut, produced full and durable remission of DM, reduced mortality, and reversed other comorbidities associated with severe obesity (eg, asthma, gastroesophageal reflux, hypertension, stress incontinence). Insulin levels decreased markedly after surgery, as did glycosylated hemoglobin (A1C) and fasting blood glucose levels. Although these effects were initially attributed to weight loss, the rapid reversal of DM within a matter of days after surgery suggest that bariatric surgery changes the signaling mechanism of the gut with pancreatic islet cells, muscles, fat, the liver, and other organs.Conclusions: Bariatric surgery has opened new vistas, producing durable full remission of type 2 DM—a breakthrough previously considered impossible—with normalization of A1C levels over time and discontinuation of all antidiabetes medication for many patients. These advances create new opportunities for exploring the mechanisms of type 2 DM and its control through pharmaceutical approaches. DM is no longer an irreversible, incurable, or hopeless disease.