Effects of age,replicative lifespan and growth rate of human nucleus pulposus cells on selecting age range for cell-based biological therapies for degenerative disc diseases

Autologous disc cell implantation, growth factors and gene therapy appear to be promising therapies for disc regeneration. Unfortunately, the replicative lifespan and growth kinetics of human nucleus pulposus (NP) cells related to host age are unclear. We investigated the potential relations among age, replicative lifespan and growth rate of NP cells, and determined the age range that is suitable for cell-based biological therapies for degenerative disc diseases. We used NP tissues classified by decade into five age groups: 30s, 40s, 50s, 60s and 70s. The mean cumulative population doubling level (PDL) and population doubling rate (PDR) of NP cells were assessed by decade. We also investigated correlations between cumulative PDL and age, and between PDR and age. The mean cumulative PDL and PDR decreased significantly in patients in their 60s. The mean cumulative PDL and PDR in the younger groups (30s, 40s and 50s) were significantly higher than those in the older groups (60s and 70s). There also were significant negative correlations between cumulative PDL and age, and between PDR and age. We found that the replicative lifespan and growth rate of human NP cells decreased with age. The replicative potential of NP cells decreased significantly in patients 60 years old and older. Young individuals less than 60 years old may be suitable candidates for NP cell-based biological therapies for treating degenerative disc diseases.     

Expression of ATP6V1C1 during oral carcinogenesis

We investigated the gene and protein expressions of V-type ATPase protein subunit C1 (ATP6V1C1) in cases of oral squamous cell carcinoma (OSCC) and contralateral normal mucosa in smokers, nonsmokers and former smokers. Subjects were separated into five groups of 15: group 1, smokers with OSCC; group 2, normal contralateral mucosa of OSCC patients; group 3, chronic smokers; group 4, former smokers who had stopped smoking 1 year earlier; group 5, individuals who had never smoked. Exfoliative cytology specimens from oral mucosa of smokers, former smokers and nonsmokers showed normal gene and protein expression. We found significantly greater gene expression in the OSCC group than in the nonsmoker groups. No difference in gene expression was observed between normal contralateral mucosa and nonsmoker groups, smoker and nonsmoker groups or former smoker and nonsmoker groups. We observed intense immunostaining for ATP6V1C1 protein in all cases of OSCC and weak or no staining in smoker, former smoker and nonsmoker groups. Significantly greater expression of ATP6V1C1 protein was observed in the OSCC group compared to the other groups, which supports the role of ATP6V1C1 in effecting changes associated with oral cancer. Analysis of the mucosae of chronic smokers, former smokers and the normal contralateral mucosa of patients with OSCC showed unaltered ATP6V1C1 gene and protein expression. Early stages of carcinogenesis, represented by altered epithelium of chronic smokers, had neither gene nor protein alterations as seen in OSCC. Therefore, we infer that the changes in ATP6V1C1 occur during later stages of carcinogenesis. Our preliminary study provides a basis for future studies of using ATP6V1C1 levels for detecting early stage OSCC.     

The isolation and preliminary characterisation of a novel Escherichia coli mutant rorB with enhanced sensitivity to ionising radiation

Summary Escherichia coli K803 cells were mutagenized and screened for the presence of clones sensitive to -rays but not to ultraviolet light. One new mutant of this type, named rorB, was isolated. This mutant is both cross-sensitive to mitomycin C and shows reduced conjugal recombination frequencies, but to a lesser extent than the phenotypically similar mutant recN. Unlike previously reported mutants of E. coli or yeast with an enhanced sensitivity to ionising radiations, rorB appears to be near wild type in ability to rejoin DNA double-strand breaks. The rorB gene maps close to ilvGEDAC at 84.5 min of the E. coli chromosome.     

Vector-mediated DNA double-strand break repair analysis in normal, and radiation-sensitive, Chinese hamster V79 cells

DNA double-strand break repair was assessed in 2 new radiation-sensitive V79 hamster cell lines (irs1 and irs2) by their ability to rejoin restriction endonuclease cuts in a transferred selectable SV40--E. coli gpt recombinant gene. The studied gene was carried in the vector pPMH16 which also contained a second selectable HSVtk-neo recombinant gene which acted as a control for DNA transformation. The parental V79 cells showed correct rejoining of KpnI and EcoRV double-strand breaks in approximately 18% and 36% of transformants respectively (correcting for the expression of undamaged gpt in neo+ transformants). irs1 shows a significantly reduced (approximately 3-fold) ability to rejoin correctly such double-strand scissions. However, irs2 rejoined such lesions as correctly as the V79 cells. The data are discussed in the context of the assay and the possible repair deficiencies of these radiosensitive mutant cells.     

Evidence from nuclear sequences that invariable sites should be considered when sequence divergence is calculated

It has long been known, from the distribution of multiple amino acid replacements, that not all amino acids of a sequence are replaceable. More recently, the phenomenon was observed at the nucleotide level in mitochondrial DNA even after allowing for different rates of transition and transversion substitutions. We have extended the search to globin gene sequences from various organisms, with the following results: (1) Nearly every data set showed evidence of invariable nucleotide positions. (2) In all data sets, substitution rates of transversions and transitions were never in the ratio of 2/1, and rarely was the ratio even constant. (3) Only rarely (e.g., the third codon position of beta hemoglobins) was it possible to fit the data set solely by making allowance for the number of invariable positions and for the relative rates of transversion and transition substitutions. (4) For one data set (the second codon position of beta hemoglobins) we were able to simulate the observed data by making the allowance in (3) and having the set of covariotides (concomitantly variable nucleotides) be small in number and be turned over in a stochastic manner with a probability that was appreciable. (5) The fit in the latter case suggests, if the assumptions are correct and at all common, that current procedures for estimating the total number of nucleotide substitutions in two genes since their divergence from their common ancestor could be low by as much as an order of magnitude. (6) The fact that only a small fraction of the nucleotide positions differ is no guarantee that one is not seriously underestimating the total amount of divergence (substitutions). (7) Most data sets are so heterogeneous in their number of transition and transversion differences that none of the current models of nucleotide substitution seem to fit them even after (a) segregation of coding from noncoding sequences and (b) splitting of the codon into three subsets by codon position. (8) These frequently occurring problems cannot be seen unless several reasonably divergent orthologous genes are examined together.      

Bayesian adaptive sequence alignment algorithms

The selection of a scoring matrix and gap penalty parameters continues to be an important problem in sequence alignment. We describe here an algorithm, the 'Bayes block aligner, which bypasses this requirement. Instead of requiring a fixed set of parameter settings, this algorithm returns the Bayesian posterior probability for the number of gaps and for the scoring matrices in any series of interest. Furthermore, instead of returning the single best alignment for the chosen parameter settings, this algorithm returns the posterior distribution of all alignments considering the full range of gapping and scoring matrices selected, weighing each in proportion to its probability based on the data. We compared the Bayes aligner with the popular Smith-Waterman algorithm with parameter settings from the literature which had been optimized for the identification of structural neighbors, and found that the Bayes aligner correctly identified more structural neighbors. In a detailed examination of the alignment of a pair of kinase and a pair of GTPase sequences, we illustrate the algorithm's potential to identify subsequences that are conserved to different degrees. In addition, this example shows that the Bayes aligner returns an alignment-free assessment of the distance between a pair of sequences.      

Synthesis of functionalized 1,8-naphthyridinones and their evaluation as novel, orally active CB1 receptor inverse agonists

Synthesis, SAR, and binding affinities are described for a new class of 1,8-naphthyridinone CB1 receptor specific inverse agonists. Food intake, knockout mouse, and pharmacokinetic evaluation of 14 indicate that this compound is an effective orally active modulator of CB1.