A multiplex PCR assay for molecular sexing of the naked mole-rat (Heterocephalus glaber)

For molecular sexing of the naked mole-rat (Heterocephalus glaber), we designed a PCR primer set to amplify part of the Y-linked DBY gene. When this primer set was applied to the samples of known sex with the 16S rRNA gene (16S rDNA) primers as control, PCR products were successfully obtained as two DNA bands in males, a male-specific 163 bp DBY band and a 446 bp band of 16S rDNA shared with females, whereas females showed only the common band. This result shows that this multiplex PCR assay is useful for sex identification of H. glaber.     

Vertebrate aging research 2006

This Hot Topics review, the first in a projected annual series, discusses those articles, published in the last year, which seem likely to have a major impact on our understanding of the aging process in mammals and the links between aging and late-life illnesses. The year's highlights include studies of oxidation damage in the very-long-lived naked mole-rat, and of caloric restriction in monkeys, humans, and growth hormone-unresponsive mice. Two studies of resveratrol, one showing its ability to extend lifespan in a short-lived fish, the other demonstrating beneficial effects in mice subjected to a diet high in fat, may well be harbingers of a parade of intervention studies in the coming decade.     

Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules

BACKGROUND: The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general. METHODS: In an attempt to explore how naked DNA enters hepatocytes the fate of a variety of molecules and particles was followed over a 24-h time frame using fluorescence microscopy. The uptake of some of these compounds was correlated with marker gene expression from a co-injected plasmid DNA. In addition, the uptake of the injected compounds was correlated with the histologic appearance of hepatocytes. RESULTS: Out of the large number of nucleic acids, peptides, proteins, inert polymers and small molecules that we tested, most were efficiently delivered into hepatocytes independently of their size and charge. Even T7 phage and highly charged DNA/protein complexes of 60-100 nm in size were able to enter the cytoplasm. In animals co-injected with an enhanced yellow fluorescent protein (EYFP) expression vector and fluorescently labeled immunoglobulin (IgG), hepatocytes flooded with large amounts of IgG appeared permanently damaged and did not express EYFP-Nuc. Hepatocytes expressing EYFP had only slight IgG uptake. In contrast, when an EYFP expression vector was co-injected with a fluorescently labeled 200-bp linear DNA fragment, both were mostly (in 91% of the observed cells) co-localized to the same hepatocytes 24 h later. CONCLUSIONS: The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery.     

The role of salicylic acid and carrot embryogenic callus extracts in somatic embryogenesis of naked oat (Avena nuda)

Enhanced somatic embryogenesis and plant regeneration have been obtained using young leaf bases of naked oat (Avena nuda) as explants by including salicylic acid (SA) and carrot embryogenic callus extracts (CECE) in media. A 5- and 4-fold improvement was achieved in somatic embryogenesis and plant regeneration on the corresponding media supplemented with 0.5 mM SA and CECE as compared to control, respectively. Some physiological and biochemical changes were assayed in both embryogenic callus (EC) and non-embryogenic callus (NEC). The results indicated that superoxide dismutase activity was stimulated and catalases and ascorbate peroxidase activities were inhibited, while the O₂ ^- (superoxide anion) content was reduced and the hydrogen peroxide level was promoted in EC compared with NEC. Reduced malondialdehyde content and relative electrolyte leakage were also detected in EC.     

In vitro regeneration of cereals based on multiple shoot induction from mature embryos in response to thidiazuron

The in vitro competency of mature cereal embryos (winter, spring and durum wheats, oat, barley and triticale) was assessed for direct multiple shoot production on culture media containing the plant growth regulators, thidiazuron (TDZ) and/or 6–benzylaminopurine (BAP). Mature embryos of CDC Dancer oat showed the best response, with 69 shoots per explant on culture medium containing a combination of 4.5 μM TDZ and 4.4 μM BAP. TDZ alone induced about 16 shoots per explant from the oat. Among the wheat genotypes, durum wheat showed the most number of shoots (35) per explant on culture medium containing 4.5 μM of TDZ and 4.4 μM of BAP. With TDZ alone, shoot regeneration for durum wheat ranged from 27–32 shoots per explant. The regeneration frequency from the three winter wheat genotypes ranged from 11–25 shoots per explant and was highest on culture medium containing 9.1 μM TDZ and 4.4 μM BAP. The latter culture medium was also effective for a triticale genotype, inducing 34 shoots per explant. The regeneration from mature embryos of barley genotypes ranged from 5–9 shoots per explant. The mature embryos of all the cereals tested could be used for in vitro regeneration with TDZ and TDZ+BAP combinations.     

Disparate patterns of age-related changes in lipid peroxidation in long-lived naked mole-rats and shorter-lived mice

A key tenet of the oxidative stress theory of aging is that levels of accrued oxidative damage increase with age. Differences in damage generation and accumulation therefore may underlie the natural variation in species longevity. We compared age-related profiles of whole-organism lipid peroxidation (urinary isoprostanes) and liver lipid damage (malondialdehyde) in long living naked mole-rats [maximum lifespan (MLS) > 28.3 years] and shorter-living CB6F1 hybrid mice (MLS approximately 3.5 years). In addition, we compared age-associated changes in liver non-heme iron to assess how intracellular conditions, which may modulate oxidative processes, are affected by aging. Surprisingly, even at a young age, concentrations of both markers of lipid peroxidation, as well as of iron, were at least twofold (P < 0.005) greater in naked mole tats than in mice. This refutes the hypothesis that prolonged naked mole-rat longevity is due to superior protection against oxidative stress. The age-related profiles of all three parameters were distinctly species specific. Rates of lipid damage generation in mice were maintained throughout adulthood, while accrued damage in old animals was twice that of young mice. In naked mole-rats, urinary isoprostane excretion declined by half with age (P < 0.001), despite increases in tissue iron (P < 0.05). Contrary to the predictions of the oxidative stress theory, lipid damage levels did not change with age in mole-rats. These data suggest that the patterns of age-related changes in levels of markers of oxidative stress are species specific, and that the pronounced longevity of naked mole-rats is independent of oxidative stress parameters.