Synergistic and threshold effects of GH1 and GHR promoter size variation on body growth and fat accrual in young Nelore (Bos indicus) bulls.

A synergistic effect in the somatotropic axis (GH1-GHR-IGF1) was observed in 736 young Nelore (Bos indicus) bulls under ad libitum grass feeding conditions on irrigated pasture in central Brazil. Stepwise substitution of shorter alleles of the promoter region of the growth hormone gene (GH1) and the P1 promoter of the GH1 receptor gene (GHR) with longer alleles was associated with significantly increased body weight gain (W550, weight at age 550 days; ADG, average daily gain) and fat accrual (FAT, rib eye fat thickness). A threshold effect on ADG was associated with allele size variation at the GH1. A best fit model indicated a 3- to 6-fold effect of GH1 variation on ADG, when compared to the variation at the GHR and a known microsatellite at the somatomedin gene (IGF1, insulin-like growth factor 1). A threshold effect on FAT was associated with substitution of the short GHR allele by the longer GHR alleles; the effect of the GHR variation on FAT was 10-fold that of the variation at the GH1 and IGF1 loci. Among the 10 GH1-GHR-IGF1 multi-genotypes identified, the predominant genotype was homozygous for the large GH1 promoter (long/long, G2/G2 or domestic type), short GHR promoter (short/short or wild type), and short IGF1 microsatellite (short/short or wild type). This predominant multi-genotype suggests that selection pressure in the Nelore breed has been directed towards high ADG and W550, and low FAT. Our results mirror previous findings in the oMtla-oGH transgenic mouse model, in which the level of somatotropic gene expression acts through a threshold mechanism, and low expression results in adipogenesis, while high expression increases body growth.

Survival of the thriftiest: restricted nurture reveals the thrifty nature of a growth gene in Bos indicus.

Growth hormone (GH) is a part of the somatotropic axis that controls metabolism, growth, development and aging in a wide range of animals. Mutations that reduce GH signaling have been associated with extended life spans and increased longevity in ways similar to what is observed in dietary restriction (DR) models. However, the mechanism by which DR works is not well understood. Here, we show that DR works as a factor in the evolution of the genetic make-up of domestic cattle. In a series of 6864 bovines of seven Bos indicus and tropically adapted Bos taurus breeds, the frequency of a short, wild-type allele of the promoter region of the bovine GH gene, G1 allele, varied from 2.7 to 17.7%. The frequency of the long, domestic G2 allele increased from 88 to 95% along 20 calf crops of commercial Bos indicus cattle of the Nelore breed undergoing selection for increasing post-weaning weight gain with ad libitum nutrient intake. Under DR, however, the G1 allele sustained growth better than the G2 allele, as observed in a series of feeding tests. The G2 allele was even detrimental or abiotropic, as it caused rapid body decay under DR. We observed a reflection symmetry of GH allele substitution effects on body weight under different dietary schemes. The G2 allele is featured as the "demanding allele", because it is optimally fitted to ad libitum nutrient intake. The G1 allele is featured as the "thrifty allele" because it is optimally fitted to DR. Our results show that dietary regimens need not extend lifespan or increase longevity in the sense of age-specific fitness. Instead, adaptation to any particular dietary regimen is just as much a consequence of selection as its cause; dietary regimens work as do any selection force, optimizing genotypic fitness to nutritional conditions.

Improving upon nature's somatic mitochondrial DNA therapies.

Mitochondrial DNA (mtDNA) directs key metabolic functions in eukaryotic cells. While a number of mtDNA mutations are known causes of human diseases and age-related dysfunctions, some mtDNA haplotypes are associated with extreme longevity. Despite the mutagenic mitochondrial environment naturally enhancing somatic mtDNA mutation rates, mtDNA remains grossly stable along generations of plant and animal species including man. This relative stability can be accounted for by the purging of deleterious mutations by natural selection operating on growing cells, tissues, organisms and populations, as observed in gametogenesis, embryogenesis, oncogenesis and cladogenesis. In the adult multicellular organism, however, mtDNA mutations accumulate in slowly dividing cells, and, to a much higher degree, in postmitotic cells and tissues. Dynamic mitochondrial fusion and fission, by redistributing polymorphic mtDNA molecules; mitophagy, by clearing defective mitochondria and mutated mtDNA; compensatory mutations and mtDNA repair can compensate for the accumulation of mtDNA mutations only to a certain extent, thereby creating a dysfunctional threshold. Here we hypothesize that this threshold is naturally up-regulated by both vertical and horizontal transfers of mtDNA from stem cells or cell types which retain the capacity of purging deleterious mtDNA through cell division and natural selection in the adult organism. When these natural cell and tissue mtDNA reserves are exhausted, artificial mtDNA therapy may provide for additional threshold up-regulation. Replacement of mtDNA has been already successfully accomplished in early stage embryos and stem cells in a number of species including primates. It is thus simply a matter of refinement of technique that somatic mtDNA therapy, i.e., therapy of pathological conditions based on the transfer of mtDNA to somatic eukaryotic cells and tissues, becomes a medical reality.

Brazilian Nelore cattle: a melting pot unfolded by molecular genetics.

The aim of the present article was to study the population structure and genetic diversity of Nelore cattle and genetic relationships between Nelore and different taurine and zebu breeds raised in Brazil. DNA polymorphism analysis was carried out with 1976 animals of 16 zebu, taurine and synthetic breeds raised in Brazil. A higher genetic differentiation was observed in taurine than in zebu cattle. Gene flow was intense between the different zebu populations. Genetic affinity analysis within the most conspicuous Brazilian zebu beef cattle, the Nelore, was carried out in a group of 615 animals from 15 representative herds. This analysis revealed at least two major Nelore subtypes, named after some genotype-phenotype associations such as the "thrifty type" and the "demanding type". This study provides molecular genetic evidence that, despite selection based on the phenotype, gene flow and gene segregation still play a major role in maintaining genetic variability within the Nelore and zebu population as a whole in Brazil.

Less DeltamtDNA4977 than normal in various types of tumors suggests that cancer cells are essentially free of this mutation

Levels of mtDNA(4977) deletions (DeltamtDNA(4977)) have been found to be lower in tumors than in adjacent non-tumoral tissues. In 87 cancer patients, DeltamtDNA(4977) was detected by multiplex polymerase chain reaction (PCR) amplification in 43 (49%) of the tumors and in 74 (85%) of the samples of non-tumoral tissues that were adjacent to the tumors. DeltamtDNA(4977) deletions were detected in 24% of the breast tumors, 52% of the colorectal tumors, 79% of the gastric tumors, and 40% of the head and neck tumors as compared with 77, 83, 100, and 90% of the adjacent respective non-tumoral tissues at the same DNA template dilution. Based on limiting dilution PCR of 16 tumors and their adjacent non-tumoral tissues, it was found that the amount of DeltamtDNA(4977) was 10- to 100-fold lower in the tumor than in the respective control non-tumoral tissues. Real-time PCR experiments were performed to quantify the number of DeltamtDNA(4977) deletions per cell, by determining the mitochondrial-to-nuclear DNA ratio. In all of the cases of breast, colorectal, gastric, and head and neck cancer the proportion of DeltamtDNA(4977) in tumors was lower than that of the respective non-tumoral tissue. Traces of DeltamtDNA(4977) in tumors were apparently due to contamination of tumor tissue with surrounding non-tumoral tissue, as evidenced by tumor microdissection and in situ PCR techniques, suggesting that tumors are essentially free of this mutation. Although the metabolic effect of DeltamtDNA(4977) may be minimal in normal (non-tumor) tissue, in tissue under stress, such as in tumors, even low levels of DeltamtDNA(4977) deletions may be intolerable.

Evaluation of a method for high yield purification of largely intact mitochondrial DNA from human placentae

We developed, and quantitatively and qualitatively evaluated an easily reproducible method for high yield purification of mitochondrial DNA (mtDNA) from human placentae by mechanical tissue disruption, differential centrifugation of mitochondria, enzymatic digestion, phenol extraction and ethanol precipitation. Average mtDNA yields were 2.5 microg/g tissue (without an RNAse treatment step) and 1.5 microg/g tissue (with an RNAse treatment step). This mtDNA migrated as a 16.5-kb isolated band in agarose gels; it yielded fragments of expected sizes after digestion with restriction enzymes; it successfully served as a template in long PCR for amplification of mtDNA sequences, and hybridized to an mtDNA probe in a predictable fashion. MtDNA yields of this method were 10-fold higher than those of previously reported ones for mtDNA purification from freshly obtained human cells and tissues, with the advantage that more placental tissue can be obtained for mtDNA purification than other types of tissue, at lower cost, and with minimal or no ethical issues

A model system for testing gene vectors using murine tumor cells on the chorioallantoic membrane of the chick embryo

We developed a model system for testing gene vectors, based on the growth of murine tumors on the chorioallantoic membrane (CAM) of embryonic chickens. The ability of selected murine cells to grow on the CAM was rated according to the following criteria: i) formation of tumor masses; ii) metastasis formation; iii) reproducibility; iv) yield, indicated as the number of embryos surviving to assessment time with visible tumors on the CAM; v) maintainability of the cell, both in the original host and the embryonic chick, or 'shuttle maintainability'; vi) detection by the naked eye, and vii) cost/benefit relation. The murine melanoma cell lineage, B16F10, which efficiently forms distinct, pigmented tumor masses and metastases on the CAM, performed better in this model than the murine B61 cell line. In vitro transduction of B16F10 cells with a recombinant adenovirus carrying a construct of the E. coli LacZ gene followed by inoculation onto the CAM resulted in beta-galactosidase expression in the tumor mass growing on the CAM. This model is potentially applicable to preclinical evaluation of gene vectors, especially for gene therapy of cancer

The challenge of vector development in gene therapy.

Gene therapy is the treatment of diseases based on the transfer of genetic information. Agents that carry or deliver DNA to target cells are called vectors (Latin vector: carrier, deliverer). Ideally, a vector should accommodate an unlimited amount of inserted DNA, lack the ability of autonomous replication of its own DNA, be easily manufactured, and be available in concentrated form. Secondly, it should have the ability to target specific cell types or to limit its gene expression to specific cell types, and to achieve sustained gene expression in the long term or in a controlled fashion. Finally, it should not be toxic or immunogenic. Such a vector does not exist and none of the DNA delivery systems so far available for in vivo gene transfer is perfect with respect to any of these points. Gene therapy and the means to promote it depend heavily on the development and improvement of new gene vector systems.

The challenge of vector development in gene therapy

Gene therapy is the treatment of diseases based on the transfer of genetic information. Agents that carry or deliver DNA to target cells are called vectors (Latin vector: carrier, deliverer). Ideally, a vector should accommodate an unlimited amount of inserted DNA, lack the ability of autonomous replication of its own DNA, be easily manufactured, and be available in concentrated form. Secondly, it should have the ability to target specific cell types or to limit its gene expression to specific cell types, and to achieve sustained gene expression in the long term or in a controlled fashion. Finally, it should not be toxic or immunogenic. Such a vector does not exist and none of the DNA delivery systems so far available for in vivo gene transfer is perfect with respect to any of these points. Gene therapy and the means to promote it depend heavily on the development and improvement of new gene vector systems

Somatic mutation analysis of the APP and Presenilin 1 and 2 genes in Alzheimer's disease brains.

The molecular basis for sporadic Alzheimer disease (AD) remains largely unknown. We hypothesized that in some cases of sporadic AD, a somatic mutation in an embryonic cell committed to neuronal development within the amyloid precursor protein (APP), the presenilin 1 (PS-1) or the presenilin 2 (PS-2) genes (genes known to be involved in familial AD) may result in AD phenotype. Using PCR, denaturing gradient gel electrophoresis (DGGE), restriction enzyme digest and direct DNA sequencing, we analyzed these genes in 99 brain tissues from patients with histopathologically proven AD. One brain sample showed a mutation within the PS-1 gene, His163 Arg, later shown to be a germline mutation. No other migration abnormalities were demonstrated in any sample in exon 16 or 17 of the APP gene or the coding exons of the PS-1 gene. Restriction digest pattern was normal with regard to the predominant PS-2 gene mutation (N141I). A known mutation in the APP gene, as well as novel mutations within the PS-1 gene were easily detected by DGGE (Reznick Wolf et al. manuscript submitted). We conclude that the genes that are involved in familial AD do not display somatic mutations in the brains of sporadic AD patients, and that other molecular mechanisms are probably involved in the pathogenesis of sporadic AD.

Degeneration in vivo of rat hippocampal neurons by wild-type Alzheimer amyloid precursor protein overexpressed by adenovirus-mediated gene transfer.

In an attempt to elucidate the pathological implications of intracellular accumulation of the amyloid precursor protein (APP) in postmitotic neurons in vivo, we transferred APP695 cDNA into rat hippocampal neurons by using a replication-defective adenovirus vector. We first improved the efficiency of adenovirus-mediated gene transfer into neurons in vivo by using hypertonic mannitol. When a beta-galactosidase-expressing recombinant adenovirus suspended in 1 M mannitol was injected into a dorsal hippocampal region, a number of neurons in remote areas were positively stained, presumably owing to increased retrograde transport of the virus. When an APP695-expressing adenovirus was injected into the same site, part of the infected neurons in the hippocampal formation underwent severe degeneration in a few days, whereas astrocytes near the injection site showed no apparent degeneration. These degenerating neurons accumulated different epitopes of APP, and beta/A4 protein (Abeta)-immunoreactive materials were undetected in the extracellular space. A small number of degenerating neurons showed nuclear DNA fragmentation. Electron microscopic examinations demonstrated that degenerating neurons had shrunken perikarya along with synaptic abnormalities. Microglial cells/macrophages were often found in close proximity to degenerating neurons, and in some cases they phagocytosed these neurons. These results suggest that intracellular accumulation of wild-type APP695 causes a specific type of neuronal degeneration in vivo in the absence of extracellular Abeta deposition.

Progressive formation of neuritic plaques and neurofibrillary tangles is exponentially related to age and neuronal size. A morphometric study of three geographically distinct series of aging people.

Neuronal size and the incidence of neuritic plaques (NPs) and neurofibrillary tangles (NFTs) were morphometrically and quantitatively studied in the entorhinal cortex of 300 autopsied individuals without dementia in three geographically distinct series (Brazil, Germany and Japan), and an additional series including 30 clinically diagnosed Alzheimer's disease patients. The mean ages at onset of NPs and NFTs were similar between the three normal series, and the incidence of NPs and NFTs increased exponentially with age, but at different rates. A correlation was found between larger neuronal size and higher incidence of NPs and NFTs. Neuronal size distribution largely seemed to account for the differences between the series. While the onset of neurodegeneration may be tightly programmed, i.e., in a species-specific manner, our data support the idea that the incidence of NPs and NFTs and the progression from NPs to NFTs may vary remarkably, depending on neuronal size.

Different rates of neuronal degeneration: an exquisite variation of the 'Cascade' hypothesis.

This morphometric, quantitative and correlative multivariate study of the hippocampal formation in human brains from two distinct normal aging populations provides an additional support to the notion that neuritic plaques (NP) are an earlier stage of the pathological process underlying neurofibrillary tangle (NFT) formation. It is shown that the rate of transformation of NP-affected neurons into NFT-bearing neurons may vary remarkably between distinct populations. In addition, it is put forward that different rates of neuronal degeneration may be one of the explanations for the existence of conflicting hypotheses regarding pathogenesis of NP and NFT and the relationships of these important histological markers to psychical deterioration.

Larger neuronal size overwhelms maintenance capacity with aging.

Neuritic plaque-affected neurons may transform into neurofibrillary tangle-bearing neurons in the aging human brain, at varying speeds depending on cell size. Smaller neurons have a greater chance to overcome the plaque stage and survive, without tangles.