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.     

Contribution of circadian physiology and sleep homeostasis to age-related changes in human sleep

The circadian pacemaker and sleep homeostasis play pivotal roles in vigilance state control. It has been hypothesized that age-related changes in the human circadian pacemaker, as well as sleep homeostatic mechanisms, contribute to the hallmarks of age-related changes in sleep, that is, earlier wake time and reduced sleep consolidation. Assessments of circadian parameters in healthy young (∼20-30 years old) and older people (∼65-75 years old)—in the absence of the confounding effects of sleep, changes in posture, and light exposure—have demonstrated that an earlier wake time in older people is accompanied by about a 1h advance of the rhythms of core body temperature and melatonin. In addition, older people wake up at an earlier circadian phase of the body temperature and plasma melatonin rhythm. The amplitude of the endogenous circadian component of the core body temperature rhythm assessed during constant routine and forced desynchrony protocols is reduced by 20-30% in older people. Recent assessments of the intrinsic period of the human circadian pacemaker in the absence of the confounding effects of light revealed no age-related reduction of this parameter in both sighted and blind individuals. Wake maintenance and sleep initiation are not markedly affected by age except that sleep latencies are longer in older people when sleep initiation is attempted in the early morning. In contrast, major age-related reductions in the consolidation and duration of sleep occur at all circadian phases. Sleep of older people is particularly disrupted when scheduled on the rising limb of the temperature rhythm, indicating that the sleep of older people is more susceptible to arousal signals genernpated by the circadian pacemaker. Sleep-homeostatic mechanisms, as assayed by the sleep-deprivation-induced increase of EEG slow-wave activity (SWA), are operative in older people, although during both baseline sleep and recovery sleep SWA in older people remains at lower levels. The internal circadian phase advance of awakening, as well as the age-related reduction in sleep consolidation, appears related to an age-related reduction in the promotion of sleep by the circadian pacemaker during the biological night in combination with a reduced homeostatic pressure for sleep. Early morning light exposure associated with this advance of awakening in older people could reinforce the advanced circadian phase. Quantification of the interaction between sleep homeostasis and circadian rhythmicity contributes to understanding age-related changes in sleep timing and quality. (Chronobiology International, 17(3), 285-311, 2000)     

Multiple origins of eusociality among sponge-dwelling shrimps (Synalpheus)

Abstract.— As the most extreme expression of apparent altruism in nature, eusociality has long posed a central paradox for behavioral and evolutionary ecology. Because eusociality has arisen rarely among animals, understanding the selective pressures important in early stages of its evolution remains elusive. Employing a historical approach to this problem, we used morphology and DNA sequences to reconstruct the phylogeny of 13 species of sponge-dwelling shrimps ( Synalpheus ) with colony organization ranging from asocial pair-bonding through eusociality. We then used phylogenetically independent contrasts to test whether sociality was associated with evidence of enhanced competitive ability, as suggested by hypotheses invoking an advantage of cooperation in crowded habitats. The molecular, morphological, and combined data each strongly supported three independent origins of monogynous, multigenerational (eusocial) colony organization within this genus. Phylogenetically independent contrasts confirmed that highly social taxa, with strong reproductive skew, have significantly higher relative abundance within the host sponge than do less social taxa, a result that was robust to uncertainty in tree topology and varying models of character change. A similar tendency for highly social species to share their sponge with fewer congener species was suggestive, but not significant. Because unoccupied habitat appears to be limiting for many sponge-dwelling shrimp species, these data are consistent with hypotheses that cooperative social groups enjoy a competitive advantage over less organized groups or individuals, where independent establishment is difficult, and that enemy pressure is of central importance in the evolution of animal sociality.     

Melanocortin-1 receptor polymorphisms and risk of melanoma: is the association explained solely by pigmentation phenotype?

Risk of cutaneous malignant melanoma (CMM) is increased in sun-exposed whites, particularly those with a pale complexion. This study was designed to investigate the relationship of the melanocortin-1 receptor (MC1R) genotype to CMM risk, controlled for pigmentation phenotype. We report the occurrence of five common MC1R variants in an Australian population-based sample of 460 individuals with familial and sporadic CMM and 399 control individuals-and their relationship to such other risk factors as skin, hair, and eye color; freckling; and nevus count. There was a strong relationship between MC1R variants and hair color and skin type. Moreover, MC1R variants were found in 72% of the individuals with CMM, whereas only 56% of the control individuals carried at least one variant (P<.001), a finding independent of strength of family history of melanoma. Three active alleles (Arg151Cys, Arg160Trp, and Asp294His), previously associated with red hair, doubled CMM risk for each additional allele carried (odds ratio 2.0; 95% confidence interval 1. 6-2.6). No such independent association could be demonstrated with the Val60Leu and Asp84Glu variants. Among pale-skinned individuals alone, this association between CMM and MC1R variants was absent, but it persisted among those reporting a medium or olive/dark complexion. We conclude that the effect that MC1R variant alleles have on CMM is partly mediated via determination of pigmentation phenotype and that these alleles may also negate the protection normally afforded by darker skin coloring in some members of this white population.     

Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes.

The CCR5-Delta32 deletion obliterates the CCR5 chemokine and the human immunodeficiency virus (HIV)-1 coreceptor on lymphoid cells, leading to strong resistance against HIV-1 infection and AIDS. A genotype survey of 4,166 individuals revealed a cline of CCR5-Delta32 allele frequencies of 0%-14% across Eurasia, whereas the variant is absent among native African, American Indian, and East Asian ethnic groups. Haplotype analysis of 192 Caucasian chromosomes revealed strong linkage disequilibrium between CCR5 and two microsatellite loci. By use of coalescence theory to interpret modern haplotype genealogy, we estimate the origin of the CCR5-Delta32-containing ancestral haplotype to be approximately 700 years ago, with an estimated range of 275-1,875 years. The geographic cline of CCR5-Delta32 frequencies and its recent emergence are consistent with a historic strong selective event (e.g. , an epidemic of a pathogen that, like HIV-1, utilizes CCR5), driving its frequency upward in ancestral Caucasian populations.     

Repeated Cocaine Administration Alters Extracellular Glutamate in the Ventral Tegmental Area

Abstract: The present study determined if repeated cocaine injections alter the effect of cocaine on extracellular glutamate in the ventral tegmental area (VTA). All rats were treated with daily cocaine (15 mg/kg i.p. × 2 days, 30 mg/kg i.p. × 5 days) or saline for 7 days. At 21 days after discontinuing the daily injections, a dialysis probe was placed into the VTA and the extracellular levels of glutamate were estimated. A systemic injection of cocaine (15 mg/kg i.p.) elevated extracellular glutamate in the VTA of rats pretreated with daily cocaine but not in the daily saline-pretreated subjects. No significant change in glutamate was produced by a saline injection in either pretreatment group. In a group of rats pretreated with daily cocaine, the D₁ antagonist SCH-23390 (30 µ M ) was infused through the dialysis probe prior to the acute injections of saline and cocaine. SCH-23390 prevented the increase in extracellular glutamate associated with the acute administration of cocaine. Behavioral data were collected simultaneously with the measures of extracellular glutamate. The behavioral stimulant effect of cocaine was greater in cocaine-pretreated than saline-pretreated subjects, and the behavioral augmentation in cocaine-pretreated rats was partly blocked by SCH-23390. These data support the hypotheses that repeated cocaine administration produces an increase in the capacity of D₁ receptor stimulation to release glutamate in the VTA and that this mechanism partly mediates behavioral sensitization produced in rats treated with daily cocaine injections.     

High levels of cathepsin B predict poor outcome in patients with breast cancer

Cathepsin B (CB) is a thiol-stimulated protease implicated in cancer invasion and metastasis. Other proteases involved in cancer spread such as urokinase-type plasminogen activator (uPA) and cathepsin D have previously been shown to be prognostic markers in breast cancer. CB was assayed by ELISA in 193 patients with primary breast cancer. CB levels were significantly higher in both primary and metastatic breast tumors than in fibroadenomas (p = 0.0001). In the primary carcinomas, CB levels showed no significant correlation with either nodal status, tumor size or estrogen receptor (ER) status. Patients with primary breast cancers containing high levels of CB had a significantly shorter disease-free interval (p = 0.01, chi-square = 6.61) and overall survival (p = 0.014, chi-square = 6.08) than patients with low levels of the protease. However, in multivariate analysis, using nodal status, tumor size, ER status and urokinase plasminogen activator (uPA), CB was not an independent prognostic marker. In contrast, nodal status, ER status and uPA were prognostic in multivariate analysis. In conclusion, CB, like certain other proteases implicated in cancer metastasis, correlates with poor outcome in patients with breast cancer. These results thus support the evidence from model systems linking CB to cancer spread. Inhibition of CB expression or activity might therefore be exploited for anti-metastatic therapies.     

Molecular evolution of P transposable elements in the genus Drosophila. III. The melanogaster species group

Phylogenetic relationships were determined for 76 partial P-element sequences from 14 species of the melanogaster species group within the Drosophila subgenus Sophophora. These results are examined in the context of the phylogeny of the species from which the sequences were isolated. Sequences from the P-element family fall into distinct subfamilies, or clades, which are often characteristic for particular species subgroups. When examined locally among closely related species, the evolution of P elements is characterized by vertical transmission, whereby the P-element phylogeny traces the species phylogeny. On a broader scale, however, the P-element phylogeny is not congruent with the species phylogeny. One feature of P-element evolution in the melanogaster group is the presence of more than one P-element subfamily, differing by as much as 36%, in the genomes of some species. Thus, P elements from several individual species are not monophyletic, and a likely explanation for the incongruence between P-element and species phylogenies is provided by the comparison of paralogous sequences. In certain instances, horizontal transfer seems to be a valid alternative explanation for lack of congruence between species and P-element phylogenies. The canonical P-element subfamily, which represents the active, autonomous transposable element, is restricted to D. melanogaster. Thus, its origin clearly lies outside of the melanogaster species group, consistent with the earlier conclusion of recent horizontal transfer.