DNA integrity and transgene expression after passage through the NOGA needle catheter used for therapeutic myocardial angiogenesis

BACKGROUND: The NOGA (Biosense Webster, Markham, ON, Canada) injection catheter is an innovative navigational device that provides an ideal platform for intra-myocardial injection material. However, injection through a long (1.91 m), narrow (27G) nitinol needle could result in deterioration in the integrity and functionality of DNA. METHODS: To test this possibility, DNA in plasmid form (pcDNA3.1) containing the Lac Z transgene (250 micro l) was passed through the NOGA needle using a hand-held 1 cc syringe at a gentle hand injection pressure (43 +/- 3 PSI, 3.0 +/- 0.2 kg/cm(2)) or at maximal manual pressure (90 +/- 6 PSI, 6.3 +/- 0.4 kg/cm(2)), either once or 20 times. This DNA, compared to DNA not passed through the NOGA needle (control), was then used to transfect primary cultures of rat skin fibroblasts (FB) from Fisher 344 rats and the cells were subsequently stained for beta galactosidase (betagal). RESULTS: Transfection efficiency was significantly reduced by passing the DNA through the needle at both 43 +/- 3 PSI (78 +/- 4% of control, n = 10, P < 0.05 versus control) and 90 +/- 6 PSI (66 +/- 4 % of control, n = 10, P < 0.01 versus control, P < 0.02 versus 43 +/- 3 PSI). Passage of the DNA through the NOGA needle 20 times resulted in a transfection efficiency of only 5 +/- 1% of control (n = 20, P < 0.1 x 10(-11) versus control). Capillary Electrophoresis revealed that the reduction in transfection efficiency was due to a conformational change in the DNA from predominantly supercoiled to nicked and linearized DNA. Transfection efficiency as compared with control decreased as the concentration of the DNA solution which was passed through the needle was increased from 0.3 micro g/ micro l to 2.4 micro g/ micro l. Recovery experiments confirmed that the reduction in transfection efficiency was not due to loss of DNA by binding to the NOGA needle. CONCLUSION: These results suggest that DNA is susceptible to shear forces when injected through the NOGA needle even at nominal clinical injection pressures, suggesting that careful and controlled injections will be required to achieve optimal gene integrity and expression.     

Mammalian ALKBH8 Possesses tRNA Methyltransferase Activity Required for the Biogenesis of Multiple Wobble Uridine Modifications Implicated in Translational Decoding

Uridines in the wobble position of tRNA are almost invariably modified. Modifications can increase the efficiency of codon reading, but they also prevent mistranslation by limiting wobbling. In mammals, several tRNAs have 5-methoxycarbonylmethyluridine (mcm⁵U) or derivatives thereof in the wobble position. Through analysis of tRNA from Alkbh8^−/− mice, we show here that ALKBH8 is a tRNA methyltransferase required for the final step in the biogenesis of mcm⁵U. We also demonstrate that the interaction of ALKBH8 with a small accessory protein, TRM112, is required to form a functional tRNA methyltransferase. Furthermore, prior ALKBH8-mediated methylation is a prerequisite for the thiolation and 2′-O-ribose methylation that form 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U) and 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm⁵Um), respectively. Despite the complete loss of all of these uridine modifications, Alkbh8^−/− mice appear normal. However, the selenocysteine-specific tRNA (tRNA^Sec) is aberrantly modified in the Alkbh8^−/− mice, and for the selenoprotein Gpx1, we indeed observed reduced recoding of the UGA stop codon to selenocysteine.tRNAs are frequently modified at the wobble uridine, a feature that is believed to either promote or restrict wobbling depending on the type of modification. In the case of eukaryotes, the functions of wobble uridine modifications have been studied in the greatest detail in Saccharomyces cerevisiae. Here, the modifications 5-methoxycarbonylmethyluridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U), and 5-carbamoylmethyluridine (ncm⁵U) or its 2′-O-ribose-methylated form, ncm⁵Um, are found in 11 out of 13 wobble uridine-containing tRNAs (22). mcm⁵U and mcm⁵s²U are mostly found in “split” codon boxes, where the pyrimidine- and purine-ending codons encode different amino acids, while ncm⁵U is found in “family” codon boxes, where all four codons encode a single amino acid. Early reports based on in vitro experiments suggested that wobble nucleosides, such as mcm⁵U, ncm⁵U, and their derivatives, may restrict wobbling (17, 37, 45), but the results of a recent comprehensive study performed in vivo in S. cerevisiae show that such modifications can improve the reading both of the cognate, A-ending codons and of the wobble, G-ending codons (22). This may suggest that the primary role of these modified nucleosides is to improve translational efficiency rather than to restrict wobbling.The characterization of wobble uridine modifications in higher eukaryotes is very limited, and little is known about the enzymes that introduce them. In mammals, mcm⁵s²U has been found in the wobble position of tRNA^Glu(UUC), tRNA^Lys(UUU), and tRNA^Arg(UCU) (40). Unlike yeast, mammals possess a specialized tRNA that is responsible for recoding the UGA stop codon to insert the 21st amino acid, selenocysteine (Sec). The mammalian tRNA^Sec population consists of two subpopulations containing either mcm⁵U or the ribose-methylated derivative mcm⁵Um in the wobble position. Interestingly, ribose methylation of mcm⁵U in tRNA^Sec appears to have a role in regulating selenoprotein synthesis, as the expression of some selenoproteins, such as glutathione peroxidase 1 (Gpx1), appears to be promoted by mcm⁵Um-containing tRNA^Sec (5, 7, 9, 32).Some years ago, the Escherichia coli AlkB protein was found to be a 2-oxoglutarate- and iron-dependent dioxygenase capable of demethylating the lesions 1-methyladenosine and 3-methylcytosine in DNA (13, 42). Multicellular organisms generally possess several different AlkB homologues (ALKBH), and bioinformatics analysis has identified eight different mammalian ALKBH proteins, denoted ALKBH1 to ALKBH8 in humans and Alkbh1 to Alkbh8 in mice, as well as the somewhat-less-related, obesity-associated FTO protein (2, 16, 30). Among the ALKBH proteins of unknown function, ALKBH8 is the only one containing additional annotated protein domains. Here, the AlkB domain is localized between an N-terminal RNA recognition motif (RRM) and a C-terminal methyltransferase (MT) domain. Interestingly, the MT domain has sequence homology to the S. cerevisiae tRNA methyltransferase Trm9, which has been shown to catalyze the methyl esterification of modified wobble uridine (U34) residues of tRNA^Arg and tRNA^Glu, resulting in the formation of mcm⁵U and mcm⁵s²U, respectively (23, 43). Until recently, human ALKBH8 was incorrectly annotated in the protein sequence database, and another human protein, KIAA1456, has been designated the human Trm9 homologue (3, 23).We have generated for this study Alkbh8-targeted mice that lack exons critical for both the MT and AlkB activities of Alkbh8. The mice did not display any overt phenotype, but tRNA from these mice was completely devoid of mcm⁵U, mcm⁵s²U, and mcm⁵Um, and the relevant tRNA isoacceptors instead contained the acid form 5-carboxymethyluridine (cm⁵U) and/or the amide forms ncm⁵U/ncm⁵s²U. Furthermore, we show that recombinant ALKBH8 and TRM112 form a heterodimeric complex capable of catalyzing the methyl esterification of cm⁵U and cm⁵s²U to mcm⁵U and mcm⁵s²U, respectively. In agreement with the involvement of mcm⁵Um in selenoprotein synthesis, we observed a reduced level of Gpx1 in the Alkbh8^−/− mice, and tRNA^Sec from these mice showed a reduced ability to decode the UGA stop codon to Sec.     

Vertebrate vitellogenin gene duplication in relation to the "3R hypothesis": correlation to the pelagic egg and the oceanic radiation of teleosts

The spiny ray-finned teleost fishes (Acanthomorpha) are the most successful group of vertebrates in terms of species diversity. Their meteoric radiation and speciation in the oceans during the late Cretaceous and Eocene epoch is unprecedented in vertebrate history, occurring in one third of the time for similar diversity to appear in the birds and mammals. The success of marine teleosts is even more remarkable considering their long freshwater ancestry, since it implies solving major physiological challenges when freely broadcasting their eggs in the hyper-osmotic conditions of seawater. Most extant marine teleosts spawn highly hydrated pelagic eggs, due to differential proteolysis of vitellogenin (Vtg)-derived yolk proteins. The maturational degradation of Vtg involves depolymerization of mainly the lipovitellin heavy chain (LvH) of one form of Vtg to generate a large pool of free amino acids (FAA 150-200 mM). This organic osmolyte pool drives hydration of the ooctye while still protected within the maternal ovary. In the present contribution, we have used Bayesian analysis to examine the evolution of vertebrate Vtg genes in relation to the "3R hypothesis" of whole genome duplication (WGD) and the functional end points of LvH degradation during oocyte maturation. We find that teleost Vtgs have experienced a post-R3 lineage-specific gene duplication to form paralogous clusters that correlate to the pelagic and benthic character of the eggs. Neo-functionalization allowed one paralogue to be proteolyzed to FAA driving hydration of the maturing oocytes, which pre-adapts them to the marine environment and causes them to float. The timing of these events matches the appearance of the Acanthomorpha in the fossil record. We discuss the significance of these adaptations in relation to ancestral physiological features, and propose that the neo-functionalization of duplicated Vtg genes was a key event in the evolution and success of the teleosts in the oceanic environment.     

Population dynamics of Salmincola salmoneus on Atlantic salmon in a northern Norwegian river

Atlantic salmon Salmo salar are often heavily infected by the gill maggot Salmincola salmoneus, but little information exists on the population dynamics of this parasite. Through a combination of in vivo field examination and laboratory analysis of gills from the Alta River S. salar population in northern Norway, we describe the population dynamics of the parasite and suggest a model for the host-parasite interactions. S. salar did not become infected with S. salmoneus until they returned to the river as first-time spawners. The infection increased rapidly until autumn, and just after spawning 96% of the spent fish (kelts) were infected with a mean intensity of 53 parasites per fish. In May, the prevalence of S. salmoneus on the descending kelts had increased to 100%, but the intensity exhibited little change. A small proportion of the adult S. salar population returned as immature to the river during autumn and had lower parasite intensities than the kelts the following spring. When the fish that had spawned previously (repeat spawners) returned from their second (or more) sea migration, they had an average infection rate of 36 S. salmoneus individuals per fish. The kelts seemed to be the main habitat for the parasite during winter and spring, and they stay long enough in the river to pass the infection to maiden S. salar that enter the river early in summer. These fish then became a source of infection for the maiden fish entering the river later. However, in years that have a possible mismatch between the opposite migration of kelts and maiden S. salar, the immature fall-running and returning repeat spawners will be crucial for maintaining the parasite population. We hypothesize that heavily infected S. salar may suffer reduced growth and survival at sea, potentially reducing the abundance of repeat spawners.     

Fluctuating asymmetry and fitness correlations in two Engraulis encrasicolus populations

Correlations among several measures of fluctuating asymmetry (FA) and fitness‐related variables were assessed in two populations of the European anchovy Engraulis encrasicolus with fast growth (Aegean Sea) and slow growth (Ionian Sea), respectively. FA levels were borderline significantly higher in the Ionian than in the Aegean for some variables. Variation in otolith shape (deviation from population norm) was lower in the Ionian than the Aegean, contrary to expectation. Within the Aegean, there was no relation between any of the FA indexes and fitness estimators, while in the Ionian a composite otolith FA index was significantly negatively correlated to standard length at age only in 2 year‐old individuals. This difference between the Aegean and Ionian may have been related to the lower growth rate in the Ionian, as FA–fitness relations may be more apparent in less‐beneficial environments. The absence of significant correlations in the Aegean and the low correlation in one age group in the Ionian suggests that FA is not a sensitive indicator of individual fitness in adult E. encrasicolus.     

Regional Gene Expression of LOX-1, VCAM-1, and ICAM-1 in Aorta of HIV-1 Transgenic Rats

Background

Increased prevalence of atherosclerotic cardiovascular disease in HIV-infected patients has been observed. The cause of this accelerated atherosclerosis is a matter of controversy. As clinical studies are complicated by a multiplicity of risk-factors and a low incidence of hard endpoints, studies in animal models could be attractive alternatives.

Methodology/Principal Findings

We evaluated gene expression of lectin-like oxidized-low-density-lipoprotein receptor-1 (LOX-1), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) in HIV-1 transgenic (HIV-1Tg) rats; these genes are all thought to play important roles in early atherogenesis. Furthermore, the plasma level of sICAM-1 was measured. We found that gene expressions of LOX-1 and VCAM-1 were higher in the aortic arch of HIV-1Tg rats compared to controls. Also, the level of sICAM-1 was elevated in the HIV-1Tg rats compared to controls, but the ICAM-1 gene expression profile did not show any differences between the groups.

Conclusions/Significance

HIV-1Tg rats have gene expression patterns indicating endothelial dysfunction and accelerated atherosclerosis in aorta, suggesting that HIV-infection per se may cause atherosclerosis. This transgenic rat model may be a very promising model for further studies of the pathophysiology behind HIV-associated cardiovascular disease.     

Phylogenetic analysis supports horizontal transfer of P transposable elements

Nucleotide sequence comparisons were used to investigate the evolution of P transposable elements and the possibility that horizontal transfer has played a role in their occurrence in natural populations of Drosophila and other Diptera. The phylogeny of P elements was examined using published sequences from eight dipteran taxa and a new, partial sequence from Scaptomyza elmoi. The results from a number of different analyses are highly consistent and reveal a P-element phylogeny that contradicts the phylogeny of the species. At least three instances of horizontal transfer are necessary to explain this incongruence, but other explanations cannot be ruled out at this time.      

Exploration of multivariate analysis in microbial coding sequence modeling

ABSTRACT: BACKGROUND: Gene finding is a complicated procedure that encapsulates algorithms for coding sequence modeling, identification of promoter regions, issues concerning overlapping genes and more. In the present study we focus on coding sequence modeling algorithms; that is, algorithms for identification and prediction of the actual coding sequences from genomic DNA. In this respect, we promote a novel multivariate method known as Canonical Powered Partial Least Squares (CPPLS) as an alternative to the commonly used Interpolated Markov model (IMM). Comparisons between the methods were performed on DNA, codon and protein sequences with highly conserved genes taken from several species with different genomic properties. RESULTS: The multivariate CPPLS approach classified coding sequence substantially better than the commonly used IMM on the same set of sequences. We also found that the use of CPPLS with codon representation gave significantly better classification results than both IMM with protein (p < 0.001) and with DNA (p < 0.001). Further, although the mean performance was similar, the variation of CPPLS performance on codon representation was significantly smaller than for IMM (p < 0.001). CONCLUSIONS: The performance of coding sequence modeling can be substantially improved by using an algorithm based on the multivariate CPPLS method applied to codon or DNA frequencies.