Neogene Research Articles

A new large didelphid of the genus Thylophorops (Mammalia: Didelphimorphia: Didelphidae), from the late Tertiary of the Pampean Region (Argentina)

We describe Thylophorops lorenzinii sp. nov. (Marsupialia, Didelphidae), the largest known didelphid opossum, living or extinct. Its type specimen comes from Late Pliocene levels at Punta San Andrés, southeastern Buenos Aires Province, Argentina. With an estimated body mass between 4.8 and 7.4 kg, it obviously surpasses that of the (up to now) largest didelphid, the living Didelphis virginiana Kerr. In addition to its larger size, the new species differs from T. chapalmalensis Ameghino and T. perplanus Ameghino in that its lower molars have more labially salient hypoconids and proportionally large hypoconulids which are not antero-posteriorly compressed.

Construction of Targeting Vector for Expressing Human GDNF in Cattle Mammary Gland

Glial cell line-derived neurotrophic factor (GDNF) is a type of neurotrophic factor with significant potential in treatment of Parkinson's disease. Combining gene targeting of animal somatic cells with nuclear transfer technique has provided a powerful method to produce transgenic animal mammary gland bioreactor. The aim of this study was to construct a gene-targeting vector for the human gdnf gene knockin at the bovine beta-casein gene locus so that human GDNF protein can be produced in the mammary gland of the gene-targeted bovine. The constructed vector contains the 2.2 kb 5' homologous arm and the 5.7 kb 3' homologous arm. The human gdnf cDNA was located at the downstream of the 5' homologous arm. The neo gene placed between the 5' and 3' homologous arms as positive selection marker gene. The HSV-tk gene and DsRed2 gene were located outside the homologous recombinant area as negative selection marker genes, respectively. The recombinant plasmids were identified by restriction fragment analysis and partial DNA sequencing. The results show that the structure of the final constructed vector accords with the designed plasmid map. In order to analyze the bioactivity of the vector, the plasmid DNA was transfected into human mammary tumor cell line Bcap-37 by lipofectamine. Reverse transcription polymerase chain reaction and Western-blotting analysis showed that the transfected cells produced human GDNF mRNA and protein. The results show that the constructed targeting vector pNRTCNbG has bioactivity to efficiently express GDNF in mammary gland cells. At the same time, it is first time to confirm that human mammary tumor cell line Bcap-37 is valid for bioactivity analysis of mammary gland specific expression vector.

Phylogeny and Biogeography of Erythronium (Liliaceae) Inferred from Chloroplast matK and Nuclear rDNA ITS Sequences

We investigated phylogenetic relationships within Erythronium using DNA sequences from the chloroplast gene matK and the internal transcribed spacer (ITS) of nuclear ribosomal DNA. Erythronium, Amana, and Tulipa form a strongly supported clade that appears to be of Asian origin. Erythronium is widely distributed in the Northern Hemisphere, and consists of three well supported and geographically distinct clades occurring in western North America, eastern North America, and Eurasia. ITS analyses suggest that the Eurasian and eastern North American clades form a monophyletic group that separated into two lineages following divergence from the western North American clade. Although the three clades are each characterized by distinct morphological features, convergence both among and within geographic areas is seen in several characters. The western North American clade exhibits greater morphological and molecular diversity than either of the other two clades, and many of its species hybridize, suggesting both recent and ongoing speciation. The geographic distribution of Erythronium suggests initial diversification in Tertiary mixed mesophytic forest, followed by fragmentation of the range during the late Tertiary or early Pleistocene and subsequent radiation in western North America.

Magnetic susceptibility of the Xifeng section and its paleoclimate significance

Magnetic susceptibility (MS) is one of the most important indicators of the East- Asia summer monsoon. Presented in this study is a high resolution magnetic susceptibility record from the Xifeng section, which consists of Quaternary loess-paleosol sequence and Pliocene red clay. Variations in magnetic susceptibility are well correlated with the δ18O record in deep-sea sediments and the eolian flux of Greenland. The climatic evolution in the Xifeng section shows that since the Late Tertiary, the driving force of the East-Asia paleo-monsoon is correlated with solar radiation on a 10-ka time scale.

Mitochondrial phylogeography of Testudo graeca in the Western Mediterranean: Old complex divergence in North Africa and recent arrival in Europe

Abstract We investigated the mitochondrial phylogeography of spur-thighed tortoises (Testudo graeca) in the Western Mediterranean. In North Africa, four major lineages (A-D) occur that together constitute a well-supported clade corresponding to one of the six major clades within T. graeca; the North African clade is sister to a Caucasian clade representing the subspecies T. g. armeniaca. Phylogenetic relationships between the North African lineages are badly resolved. Lineage A is distributed in Tunisia and adjacent Algeria, lineage B in Algeria and northern Morocco, lineage C in the Libyan Cyrenaica Peninsula, and lineage D north of the High Atlas Mts. and in the Souss Valley (southern Morocco). Lineage B is subdivided into two subgroups, B1 (eastern Morocco and Algeria) and B2 (north-western Morocco). Italian tortoises harbour haplotypes of lineage A, Spanish tortoises of subgroup B1. Based on a relaxed molecular clock calibrated with fossil evidence, the six major mtDNA clades of T. graeca are estimated to have diverged approximately 4.2-1.8 Ma ago; the split between the clades representing the eastern subspecies T. g. ibera and T. g. terrestris is younger than the split between Western Mediterranean tortoises and T. g. armeniaca. The Western Mediterranean lineages A-D were dated to have diverged at least 1.4-1.1 Ma ago; B1 and B2 split approximately 0.7 Ma ago. Our results suggest that Italian and Spanish tortoises were either introduced or originated from trans-oceanic dispersal in historic or prehistoric times. Spur-thighed tortoises invaded North Africa probably across Near Eastern landbridges that emerged in the Late Tertiary. Their diversification in North Africa seems to be correlated with habitat aridization cycles during the Pleistocene. The ranges of the Western Mediterranean lineages largely correspond to the distribution of morphologically defined subspecies in North Africa, with exception of T. g. graeca and T. g. whitei, and of T. g. lamberti and T. g. marokkensis, which are not differentiated. We propose to lump the first two subspecies under the name of T. g. graeca and the latter under the name of T. g. marokkensis. The complex differentiation of spur-thighed tortoises in North Africa implies that the model of a bipartite east-west differentiation, as proposed for other Maghrebian amphibians and reptiles, may be too simplistic, reflecting incomplete locality sampling rather than actual phylogeographic differentiation.

Why Were Extinct Gigantic Birds So Small?

This review details the six lineages of large flightless birds that evolved in the Late Cretaceous, Tertiary and Quaternary periods of geological time. Estimates of mass for each type of bird suggest that maximal mass is no greater than 500kg with most species attaining only 250–300 kg or less. By contrast, non-avian Archosaurs of the Mesozoic, and many mammal species of the Tertiary, attained great size with many species reaching several tonnes. Size has been limited in flightless birds because of the strength of the eggshell and in the largest species reproduction was only possible if the smaller males incubated. That reproductive characteristics limit mass in flightless birds suggests that truly gigantic non-avian theropods could not contact incubate their eggs and had to rely on environmental sources of heat energy to drive embryonic development. If fossil evidence ever arises to support proper contact incubation in a non-avian theropod then it is predicted that it will only be from a small (<250 kg) species.

Effect of wt p53 gene transfection on sensitivity of hyperthermia in C6 glioma cells

To investigate the effect of wt p53 gene transfection on the sensitivity of hyperthermia in C6 glioma. The recombinant eukaryotic expressive vector pCMV-p53 plasmids were extracted, identified and transmitted to C6 glioma cells by stable transfection. The biological characteristics were compared among C6/p53 (+) cells, thermo-treated C6/p53 (+) cells, thermo-treated C6 cells, thermotolerance C6 cells and control C6 cells. Transplanted glioma was treated by laser interstitial thermotherapy (LITT) in tumor-bearing nude mice. The wt p53 gene segments were extracted and correctly identified by restriction enzyme (Hind and EcoR). The optimal transfection concentration of plasmid to liposome was 1:6, neo gene expressed stably in C6 cells transfected with positive and blank plasmid. Thermotolerance C6 cell growth was normal in vitro, growth of C6/p53 (+) cells was inhibited. Growth of hyperthermia treated C6 cells was inhibited significantly 12 h after thermal treatment, cell proliferation activity of C6/p53 (+) cells was suppressed significantly 6 h after hyperthermia treatment. Apoptosis was observed in C6/p53 (+), thermo-treat C6 and thermo-treat C6/p53 (+) cells. The apoptosis ratio of hyperthermia treated C6/p53(+) cells was increased by 30 folds compared with that of control groups. Study in nude mouse model demonstrated lower positive inoculation rate in groups of C6/p53 (+) cells (57 %), hyperthermia treated C6 cells (75 %) and hyperthermia treated C6/p53(+) cells (20 %). C6/p53(+) LITT group showed the highest antitumor effect, compared with C6 LITT group(P<0.05). However, thermotolerance C6 LITT group had the lowest effect. Transfection of wt p53 gene can increase the sensitivity of hyperthermia in glioma cells.

Disruption of the meprin α and β genes in mice alters homeostasis of monocytes and natural killer cells

Meprin metalloproteases are implicated in inflammatory bowel disease, which involves dysfunction of immune cells. However, the roles of meprins in the immune and hematological system remain uncharacterized. In this report, we demonstrate that meprins were expressed in the hematological system, and meprin alpha/beta null (alpha(-/-)/beta(-/-)) mice had decreased prevalence of resident monocytes and natural killer (NK) cells in blood, with a concomitant accumulation of inflammatory monocytes and NK cells in bone marrow. In contrast, T and B lymphocytes were not affected by meprin deficiency. In response to acute inflammation induced by intraperitoneal injection of thioglycollate, meprin-deficient mice exhibited higher body temperature than wild-type mice, which was correlated with retention of inflammatory monocytes, but persistent low prevalence of NK cells in blood. These results indicate that meprin metalloproteases play important roles in the homeostasis of monocytes and NK cells, and possibly are involved in egress of these two type cells from bone marrow and homing to the periphery. Our findings are the first report to demonstrate that metalloproteases affect homeostasis of leukocytes, which have important implications for understanding physiology of and pathogenesis in the hematological system.

Systematics and Zoogeography of the Rock Basses (Centrarchidae: Ambloplites)

This study examines the role that historical events have played in the diversification of members of the genus Ambloplites by estimating divergence times of clades within the genus. DNA sequences of the mitochondrial cytochrome b gene were used to develop phylogenetic hypotheses for Ambloplites using maximum parsimony and Bayesian methods. The timing of diversification within and between species of Ambloplites was estimated using nonparametric rate-smoothing and penalized likelihood methods calibrated by fossil dates. Monophyly of three of the four species was supported. The ages of the most recent common ancestors of species of Ambloplites estimated in this study confirm earlier estimates that in most instances, their origins occurred prior to the Pleistocene. A comparison of estimated divergence times of lineages to sea level fluctuations indicates a correspondence to extremely high or low sea stands throughout the Neogene Period. Populations of Ambloplites in the Ozarks and Ouachita highlands previously assigned to A. ariommus appear to have either been introgressed by A. rupestris or were misidentified as A. ariommus. Ambloplites rupestris displays low genetic divergence throughout its range and is consistent with a re-colonization of previously glaciated areas following the Pleistocene.

THE BIOGEOGRAPHIC ORIGIN OF ARCTIC ENDEMIC SEAWEEDS: A THERMOGEOGRAPHIC VIEW(1).

The Arctic is geologically and biogeographically young, and the origin of its seaweed flora has been widely debated. The Arctic littoral biogeographic region dates from the latest Tertiary and Pleistocene. Following the opening of Bering Strait, about 3.5 mya, the "Great Trans-Arctic Biotic Interchange" populated the Arctic with a fauna strongly dominated by species of North Pacific origin. The Thermogeographic Model (TM) demonstrates why climate and geography continued to support this pattern in the Pleistocene. Thus, Arctic and Atlantic subarctic species of seaweeds are likely to be evolutionarily "based" in the North Pacific, subarctic species are likely to be widespread in the warmer Arctic, and species of Atlantic Boreal or warmer origin are unlikely in the Arctic and Subarctic. Although Arctic seaweeds have been thought to have a greater affinity with the North Atlantic, we have reanalyzed the Arctic endemic algal flora, using the Thermogeographic Model and evolutionary trees based on molecular data, to demonstrate otherwise. There are 35 congeneric species of the six, abundant Arctic Rhodophyta that we treat in this paper; 32 of these species (91%) occur in the North Pacific, two species (6%) occur in the Boreal or warmer Atlantic Ocean, and a single species is panoceanic, but restricted to the Subarctic. Laminaria solidungula J. Agardh, a kelp Arctic "endemic" species, has 18 sister species. While only eleven (61%) occur in the North Pacific, this rapidly dispersing and evolving genus is a terminal member of a diverse family and order (Laminariales) widely accepted to have evolved in the North Pacific. Thus, both the physical/time-based TM and the dominant biogeographic pattern of relatives of Arctic macrophytes suggest strong compliance with the evidence of zoology, geology, and paleoclimatology that the Arctic marine flora is largely of Pacific origin.

The Identification of Genes Cooperating with Mpl Signaling Using Insertional Mutagenesis

In addition to its role in megakaryopoiesis and platelet production, thrombopoietin activation of its receptor, myeloproliferative leukemia virus protooncogene (Mpl), results in hematopoietic stem cell (HSC) homeostasis and self-renewal. Mutations in the Mpl gene have been shown to be associated with myeloproliferative disorders. In an attempt to identify genes that could cooperate with Mpl signaling leading to an increase in cell survival and proliferation, we have used retroviral expression of a drug dependent, dimerizable, fusion protein which contains the cytoplasmic domain of Mpl. In previous experiments the expression of this protein in both mouse and human blood progenitors resulted in drug dependent hematopoietic cell expansion. We cloned a bacterial shuttle plasmid carrying the Neo gene and a bacterial origin of replication into the 3′ untranslated region of the well-described MSCV based retroviral vector MGIFM to produce the MGIFMNO vector. The shuttle plasmid allows nonbiased recovery of provirus genomic integration sites. This vector can interrupt gene structure through insertion, and has an intact long terminal repeat which can activate adjacent genes. We transduced the human leukemia cell line K562 with the MGIFMNO vector carrying inducible Mpl fusion construct and bacterial shuttle plasmid. The transduction efficiency and relative Mpl expression were assayed by GFP expression since GFP and the Mpl fusion gene are co-expressed through an internal ribosome entry site (IRES). After transduction, the endogenous, transforming Bcr-Abl kinase was blocked using imatinib, and cells dependent on Mpl signaling were selected by the addition of the dimerizer drug, AP20187. In the absence of Mpl signaling the cells underwent erythroid differentiation and died. A small proportion of the transduced cells (3%) survived in the presence of AP20187. The mean fluorescent intensity of the surviving cells was increased relative to non-selected, transduced cells indicating that selection required a high level of Mpl expression. The surviving cells in long term culture were dependent on Mpl signaling and were sensitive to a Jak2 inhibitor, AG490. We cloned retroviral integration sites from the established Mpl dependent population using the plasmid rescue method. A scoring system to assign relevance to isolated integration sites was developed, based on:multiple independent recoveries,relative distance from a target gene, andproximity to a target gene that encodes a product involved in cell cycle, apoptosis, senescence, or gene expression regulation.Cells that have a proliferative advantage and are dependent on Mpl signaling should contain synergistic mutations. Insertion sites in genomic regions appearing repeatedly in multiple experiments are considered highly significant. A preliminary screen of 46 positive integration clones revealed 17 independent integration sites, confirming feasibility of the screening method. Within the rescued integrations we identified possible candidate genes including:the RSL1D1 gene, also labeled PBK1 – nucleostemin interacting protein (nucleostemin was suggested to regulate HSC proliferation),the acidic nuclear phosphoprotein 32 family member B – cell cycle and survival factor,the SH3-domain kinase binding protein 1 – Cbl interacting protein (Cbl was indicated in a repression of HSC self renewal),and Angiotensin II receptor-associated protein isoform E.Large scale screening of integration sites and experimental analysis of candidate genes are under way.

Carving the History of East Asia′s East-Tilting Topography and East Asian Monsoon——An Introduction to IODP Proposal 683

Ocean drilling in the Bengal Fan has revealed the uplift history of the Himalayas and Indian monsoon,but there is no analogous deep-water fan in the Western Pacific marginal seas that can be drilled and used to constrain the Tibet uplift history from its eastern side.The Yangtze river,originating from northeastern Tibet and draining a large area prevailed by monsoon precipitation,is highly sensitive to plateau uplift and monsoon evolution.A systematical study of the Yangtze river deposits will test various hypotheses concerning plateau uplift and its link with monsoon evolution.Along the modern Yangtze river,the upper reach is decoupled from the lower reach by the Three Gorges.About 30% of the total sediment load from the Yangtze river is laid in the Jianghan Basin immediately out from the Gorges,~40% is deposited in the lower reach and delta,and only ~30% is transported to the East China Sea.Therefore,a combination of ocean and continental drilling along the Yangtze River will recover records useful for carving the history of uplift and monsoon.A close timing of tectonic and climatic events mainly at four time intervals has been proposed as evidence for a link between Tibet uplift and the Asian monsoon initiation: the late Pliocene(2~3 Ma),the late Miocene(~8 Ma),the early-middle Miocene(~15 Ma) and the latest Oligocene(~25 Ma).On the basis of the Three Gorges incision or delta development data,however,the history of the Yangtze River can be traced back only to the late Pliocene or early Pleistocene,and the East China Sea came into existence only in the latest Miocene.A younger Yangtze river is in a sharp contrast to the early uplift of Tibet.In the Paleogene,a broad belt of aridity stretched across China from west to east,and numerous lake basins developed in rift grabens,prohibiting any east-flowing large rivers.Large rivers like the Yangtze river could have developed only since Neogene with the advent of an east-tilting topography and monsoon in East Asia.The proposed joint IODP/ICDP project will provide sediment records from marine and terrestrial basins to help clarify the most puzzling issues in East Asian environmental evolution and to test the hypothetical link between Tibetan uplift and monsoon climate from the eastern side of the plateau.

Correlation of Himalayan exhumation rates and Asian monsoon intensity

Although most data suggest that the India–Eurasia continental collision began ∼45–55 Myr ago, the architecture of the Himalayan–Tibetan orogen is dominated by deformational structures developed in the Neogene period (<23 Myr ago). The stratigraphic record and thermochronometric data indicate that erosion of the Himalaya intensified as this constructional phase began and reached a peak around 15 Myr ago. It remained high until ∼10.5 Myr ago and subsequently slowed gradually to ∼3.5 Myr ago, but then began to increase once again in the Late Pliocene and Pleistocene epochs. Here we present weathering records from the South China Sea, Bay of Bengal and Arabian Sea that permit Asian monsoon climate to be reconstructed back to the earliest Neogene. These indicate a correlation between the rate of Himalayan exhumation—as inferred from published thermochronometric data—and monsoon intensity over the past 23 Myr. We interpret this correlation as indicating dynamic coupling between Neogene climate and both erosion and deformation in the Himalaya. Although the India–Eurasia collision initiated ∼50 Myr ago, major deformation and exhumation of the Himalaya did not begin until the early Neogene (∼23 Myr ago). This coincides with the increased intensity of the Asian monsoons, as indicated by weathering records from the South China Sea, Bay of Bengal and Arabian Sea, and hints at a dynamic coupling between climate and both erosion and deformation in the Himalaya.

The Strait of Gibraltar as a melting pot for plant biodiversity

The S Iberian Peninsula and NW Africa constitute one of the main hotspots for plant biodiversity within the Mediterranean Basin. At the core of this hotspot, across the Strait of Gibraltar, lies a smaller region whose singular Cenozoic history and ecological features have created a distinct, nested sub-hotspot. In particular, an important relict flora, a high endemism, and the dominance of vegetation types other than the paradigmatic Mediterranean-type sclerophyllous forests and shrublands, are biogeographical features of paramount relevance. These singular environments may have provided suitable mesic habitats for stable human populations in this region throughout the Quaternary. Here we explore the possible causes of the unique biodiversity of this region. First, we show that endemism is associated with poor soils and mild Mediterranean climate, whereas relictness is primarily associated with riparian and humid habitats which might have remained relatively stable since the Late Tertiary. We then review and provide new results on lineage and molecular diversity of some taxa based on their evolutionary relationships inferred from phylogenetic and phylogeographical analyses. These indicate that long-lived plants, such as trees, tend to show some discontinuity in effective gene flow, in comparison with short-lived and pioneer plants. Colonization abilities rather than dispersal vectors appear to be a crucial factor. We also analyze the role of microevolutionary processes that generate plant biodiversity in terms of reproductive features involved in lineage formation, such as breeding system, pollination biology and postdispersal regeneration. The few available studies show that this region is prone to changes in reproductive systems, associated with both abiotic and biotic conditions in the new habitats that plants face during colonization. Dispersal and regeneration ecology is especially important for relict species, as most of these species face some biotic (e.g., lack of dispersers, predation) and abiotic (edaphic and/or microclimate requirements) limitations to their recruitment. Nevertheless current limitations in the recruitment of relict plant species should not obscure the fact that they have been resilient in the face of Plio–Pleistocene climate changes in the Gibraltar region, whereas they became extinct in other Mediterranean areas. The relatively stable and mild climate of this region may have provided a suitable long-term environment for many other organisms, including humans.

Down‐regulation of protocadherin‐α A isoforms in mice changes contextual fear conditioning and spatial working memory

Diverse protocadherins (Pcdhs), which are encoded as a large cluster (composed of alpha, beta and gamma clusters) in the genome, are localized to axons and synapses. The Pcdhs have been proposed to contribute to the generation of sophisticated neural networks and to regulate brain function. To address the molecular roles of Pcdhs in regulating individual behavior, here we generated knockdown mice of Pcdh-alpha proteins and examined their behavioral abnormalities. There are two alternative splicing variants of the Pcdh-alpha constant region, Pcdh-alpha A and B isoforms, with different cytoplasmic tails. Pcdh-alpha(DeltaBneo/DeltaBneo) mice, in which the Pcdh-alpha B splicing variant was absent and the Pcdh-alpha A isoforms were down-regulated to approximately 20% of the wild-type level, exhibited enhanced contextual fear conditioning and disparities in an eight-arm radial maze. Similar abnormalities were found in Pcdh-alpha(DeltaAneo/DeltaAneo) mice, which lacked 57 amino acids of the Pcdh-alpha A cytoplasmic tail. These learning abnormalities were, however, not seen in Pcdh-alpha(DeltaB/DeltaB) mice [in which the neomycin-resistance (neo) gene cassette was removed from the Pcdh-alpha(DeltaBneo/DeltaBneo) alleles], in which the expression level of the Pcdh-alpha A isoforms was recovered, although the Pcdh-alpha B isoforms were still completely missing in the brain. In addition, the amount of 5-hydroxytryptamine increased in the hippocampus of the hypomorphic Pcdh-alpha A mutant mice but not in recovery Pcdh-alpha(DeltaB/DeltaB). These results suggested that the level of Pcdh-alpha A isoforms in the brain has an important role in regulating learning and memory functions and the amount of 5-hydroxytryptamine in the hippocampus.

Deformation structures developed in the Neogene strata in Dewa hilly land, Akita Prefecture

出羽丘陵は，東北日本が強い東西圧縮応力場におかれた鮮新世前期から逆断層や褶曲構造を形成しながら隆起を開始し，更新世中期にかけて全域が陸化したと考えられている．この隆起運動に関連した変形構造は，出羽丘陵成立過程の構造運動の実態を明らかにするために重要なデータとなるだけでなく，脆性領域の変形機構や変形様式を理解するための格好の素材を提供している．この見学旅行では，出羽丘陵西縁部に存在する北由利衝上断層群および中央部に位置する鳥田目断層群に伴われる変形構造を観察し，その運動像や変形条件について考察する．

High efficiency transformation of Tetrahymena using a codon-optimized neomycin resistance gene

Tetrahymena thermophila is a useful model for the study of eukaryotic biology. A neomycin resistance gene ( neo) has been developed that was optimized for the codon usage of T. thermophila. Using this codon-optimized neo gene ( neoTet), a new drug resistance marker cassette, neo4, has been constructed. The neo4 cassette resulted in about ten times more drug resistant transformants than a cassette containing the non-codon-optimized original neo gene. The new cassette enables transgenic Tetrahymena strains to be created with high efficiency. This study also emphasizes the importance of codon optimization in transgene expression in Tetrahymena.

Radioprotection In Vitro and In Vivo by Minicircle Plasmid Carrying the Human Manganese Superoxide Dismutase Transgene

Manganese superoxide dismutase plasmid liposomes (MnSOD-PL) confer organ-specific in vivo ionizing irradiation protection. To prepare for potential intravenous clinical trials of systemic MnSOD-PL for radioprotection in humans, plasmid and bacterial sequences were removed and a new minicircle construct was tested. Minicircle MnSOD was purified and then cotransfected into 32D cl 3 murine interleukin-3-dependent hematopoietic progenitor cells along with another plasmid carrying the neo gene. Cells were selected in G418 (50 microg/ml) and cloned by limiting dilution. Biochemical analysis of minicircle MnSOD-transfected cells showed an MnSOD biochemical activity level of 5.8 +/- 0.5 U/mg compared with 2.7 +/- 0.1 U/mg for control 32D cl 3 cells (p = 0.0039). 32D-mc-MnSOD cells were as radioresistant as full-length MnSOD-PL transgene-expressing 2C6 cells, relative to 32D cl 3 parent cells, with an increased shoulder on the radiation survival curve (n = 4.8 +/- 0.2 and n = 4.6 +/- 0.2, respectively, compared with 1.5 +/- 0.5 for 32D cl 3 cells; p = 0.007). C57BL/6NHsd mice received intraoral mc-MnSOD-PL, mc-DsRed-PL control, full-length MnSOD-PL, or blank-PL and then were irradiated 24 hr later with 31 Gy to the esophagus. Mice receiving mc-MnSOD-PL showed increased survival compared with control mice or mice treated with mc-DsRed-PL (p = 0.0003 and 0.039, respectively), and comparable to full-length MnSOD-PL. Intravenous, systemic administration of mc-MnSOD-PL protected mice from total body irradiation (9.75 Gy). Therefore, minicircle DNA containing the human MnSOD transgene confers undiminished radioprotection in vitro and in vivo.

The case for the original Neogene

The case for the original Neogene

Radioprotection In Vitro and In Vivo by Mini Circle Plasmid Containing the Human Manganese Superoxide Dismutase (MnSOD) Transgene

Manganese superoxide dismutase-plasmid liposomes (MnSOD-PL) confer organ specific in vivo ionizing irradiation protection. To prepare for potential intravenous clinical trials of systemic MnSOD-PL for radioprotection in humans, plasmid and bacterial sequences were removed and a new mini circle construct was tested. Mini circle-MnSOD was purified and then co-transfected into 32D cl 3 murine IL-3 dependent hematopoietic progenitor cells along with another plasmid containing the neo gene. Cells were selected in G418 (50 μg/ml) and cloned by limiting dilution. Biochemical analysis of mini circle MnSOD transfected cells showed 5.8 ± 0.5 U/mg MnSOD biochemical activity compared to 2.7 ± 0.1 U/mg for control 32D cl 3 cells (p = 0.0039). 32D -MC-MnSOD cells were comparably radioresistant to full length MnSOD-PL transgene 2C6 cells, relative to 32D cl 3 parent cells, with an increased shoulder on the radiation survival curve (n = 4.8 ± 0.2 and n = 4.6 ± 0.2 respectively compared to 1.5 ± 0.5 for 32D cl 3, p = 0.007). C57BL/6NHsd mice received intra-oral MC-MnSOD-PL, MC-DS-red-PL control, full length MnSOD-PL, or blank-PL then were irradiated 24 hours later to 31 Gy to the esophagus. Mice receiving MC-MnSOD-PL showed increased survival compared to control mice or mice treated with MC-DS-red-PL (p = 0.0099, or 0.039, respectively), and comparable to full length MnSOD-PL. Intravenous, systemic administration of MC-MnSOD-PL protected mice from 9.75 Gy total body irradiation. Therefore, mini circle DNA containing the human MnSOD transgene confers undiminished radioprotection in vitro and in vivo.