Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene.

A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled transactivator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament 200 kDa, and glutamic acid decarboxylase in accordance with the reduced production of the v-myc oncoprotein. Differentiated HC2S2 cells expressed large sodium and calcium currents and could fire regenerative action potentials. These results suggest that the suppression of the v-myc oncogene may be sufficient to make proliferating cells exit from cell cycles and induce terminal differentiation. The HC2S2 cells will be valuable for studying the differentiation process of neurons.

Glial cell line-derived neurotrophic factor but not transforming growth factor beta 3 prevents delayed degeneration of nigral dopaminergic neurons following striatal 6-hydroxydopamine lesion.

Glial cell line-derived neurotrophic factor (GDNF) and transforming growth factor beta 3 (TGF-beta 3) are members of the TGF-beta superfamily with high neurotrophic activity on cultured nigral dopamine neurons. We investigated the effects of intracerebral administration of GDNF and TGF-beta 3 on the delayed cell death of the dopamine neurons in the rat substantia nigra following 6-hydroxydopamine lesions of dopaminergic terminals in the striatum. Fluorescent retrograde tracer injections and tyrosine hydroxylase immunocytochemistry demonstrated nigral degeneration with an onset 1 week after lesion, leading to extensive death of nigral neurons 4 weeks postlesion. Administration of recombinant human GDNF for 4 weeks over the substantia nigra at a cumulative dose of 140 micrograms, starting on the day of lesion, completely prevented nigral cell death and atrophy, while a single injection of 10 micrograms 1 week postlesion had a partially protective effect. Continuous administration of TGF-beta 3, starting on the day of lesion surgery, did not affect nigral cell death or atrophy. These findings support the notion that GDNF, but not TGF-beta 3, is a potent neurotrophic factor for nigral dopamine neurons in vivo.

WW6: an embryonic stem cell line with an inert genetic marker that can be traced in chimeras.

Mutant mice produced by gene targeting in embryonic stem (ES) cells often have a complex or embryonic lethal phenotype. In these cases, it would be helpful to identify tissues and cell types first affected in mutant embryos by following the contribution to chimeras of ES cells homozygous for the mutant allele. Although a number of strategies for following ES cell development in vivo have been reported, each has limitations that preclude its general application. In this paper, we describe ES cell lines that can be tracked to every nucleated cell type in chimeras at all developmental stages. These lines were derived from blastocysts of mice that carry an 11-Mb beta-globin transgene on chromosome 3. The transgene is readily detected by DNA in situ hybridization, providing an inert, nuclear-localized marker whose presence is not affected by transcriptional or translational controls. The "WW" series of ES lines possess the essential features of previously described ES lines, including giving rise to a preponderance of male chimeras, all of which have to date exhibited germ-line transmission. In addition, clones selected for single or double targeting events form strong chimeras, demonstrating the feasibility of using WW6 cells to identify phenotypes associated with the creation of a null mutant.

Generation of targeted retroviral vectors by using single-chain variable fragment: an approach to in vivo gene delivery.

We report the generation of a retroviral vector that infects human cells specifically through recognition of the low density lipoprotein receptor. The rationale for this targeted infection is to add onto the ecotropic envelope protein of Moloney murine leukemia virus, normally trophic for murine cells, a single-chain variable fragment derived from a monoclonal antibody recognizing the human low density lipoprotein receptor. This chimeric envelope protein was used to construct a packaging cell line producing a retroviral vector capable of high-efficiency transfer of the Escherichia coli beta-galactosidase gene to human cells expressing low density lipoprotein receptor. This approach offers a generalized plan to generate cell and tissue-specific retroviral vectors, an essential step toward in vivo gene therapy strategies.

Immunoglobulin variable region hypermutation in hybrids derived from a pre-B- and a myeloma cell line.

Somatic mutation of the variable (V) regions of immunoglobulin genes occurs in vivo at rates that have been estimated to be between 10(-3) and 10(-4) per bp per generation. To study this process in vitro, the 18.81 pre-B-cell line and hybrids derived by fusing 18.81 to the NSO myeloma fusion partner were transfected with a mu heavy-chain construct containing a nonsense mutation in the V region (Vn) or the constant region (Cn). Mutation was quantitated by reversion analysis using the ELISA spot assay to detect single cells secreting IgM. Fluctuation analysis revealed that V-region mutations spontaneously occurred in 18.81 cells at an average rate of 5.8 x 10(-6) per bp per cell generation and in selected 18.81-NSO hybrids at greatly increased rates of 1.6 x 10(-3) to 5.8 x 10(-4) per bp per generation. The Vn construct also reverted frequently in transgenic mice, indicating that it contained sufficient information to mutate at high rates both in vivo and in vitro. Sequence analysis of reverted genes revealed that reversion was due to point mutations. Since the rates and nature of the mutations that are occurring in these transfected genes are similar to those reported in vivo, it should be possible to use this system to identify the cis-acting sequences and trans-acting factors that are responsible for V-region somatic hypermutation.

Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries.

Fusion phage libraries expressing single-chain Fv antibodies were constructed from the peripheral blood lymphocytes of two melanoma patients who had been immunized with autologous melanoma cells transduced the gamma-interferon gene to enhance immunogenicity, in a trial conducted at another institution. Anti-melanoma antibodies were selected from each library by panning the phage against live cultures of the autologous tumor. After two or three rounds of panning, clones of the phage were tested by ELISA for binding to the autologous tumor cells; > 90% of the clones tested showed a strong ELISA reaction, demonstrating the effectiveness of the panning procedure for selecting antimelanoma antibodies. The panned phage population was extensively absorbed against normal melanocytes to enrich for antibodies that react with melanoma cells but not with melanocytes. The unabsorbed phage were cloned, and the specificities of the expressed antibodies were individually tested by ELISA with a panel of cultured human cells. The first tests were done with normal endothelial and fibroblast cells to identify antibodies that do not react, or react weakly, with two normal cell types, indicating some degree of specificity for melanoma cells. The proportion of phage clones expressing such antibodies was approximately 1%. Those phage were further tested by ELISA with melanocytes, several melanoma lines, and eight other tumor lines, including a glioma line derived from glial cells that share a common lineage with melanocytes. The ELISA tests identified three classes of anti-melanoma antibodies, as follows: (i) a melanoma-specific class that reacts almost exclusively with the melanoma lines; (ii) a tumor-specific class that reacts with melanoma and other tumor lines but does not react with the normal melanocyte, endothelial and fibroblast cells; and (iii) a lineage-specific class that reacts with the melanoma lines, melanocytes, and the glioma line but does not react with the other lines. These are rare classes from the immunized patients' repertoires of anti-melanoma antibodies, most of which are relatively nonspecific anti-self antibodies. The melanoma-specific class was isolated from one patient, and the lineage-specific class was isolated from the other patient, indicating that different patients can have markedly different responses to the same immunization protocol. The procedures described here can be used to screen the antibody repertoire of any person with cancer, providing access to an enormous untapped pool of human monoclonal anti-tumor antibodies with clinical and research potential.

Optical and ultraviolet observations of a strong flare in the young, single K2 dwarf LQ Hya

We present high-resolution optical echelle spectra and IUE observations during a strong flare on 1993 December 22 in the very active, young, rapidly rotating, single K2 dwarf LQ Hya. The initial impulsive phase of the flare, which started sometime between 2:42 ut and 4:07 ut, was characterized by strong optical continuum enhancement and blueshifted emission lines with broad wings. The optical chromospheric lines reached their maximum intensity at ≈ 5:31 ut, by which time the blueshift vanished and the optical continuum enhancement had sharply decreased. Thereafter, the line emission slowly decreased and the lines redshift in a gradual phase that lasted at least two more hours. The Mg II lines behaved similarly. Quiescent C IV flux levels were not recovered until 21 h later, though a data gap and a possible second flare make the interpretation uncertain. In addition to the typically flare-enhanced emission lines (e.g., H α and H β), we observe He I D_3 going into emission, plus excess emission (after subtraction of the quiescent spectrum) in other He I and several strong neutral metal lines (e.g., Mg I b). Flare enhancement of the far-ultraviolet continuum generally agrees with an Si I recombination model. We estimate the total flare energy, and discuss the broad components, asymmetries and Doppler shifts seen in some of the emission lines.

Theory of single-spin inelastic tunneling spectroscopy

I show that recent experiments of inelastic scanning tunneling spectroscopy of single and a few magnetic atoms are modeled with a phenomenological spin-assisted tunneling Hamiltonian so that the inelastic dI/dV line shape is related to the spin spectral weight of the magnetic atom. This accounts for the spin selection rules and dI/dV spectra observed experimentally for single Fe and Mn atoms deposited on Cu2N. In the case of chains of Mn atoms it is found necessary to include both first and second-neighbor exchange interactions as well as single-ion anisotropy.

A Hardware-Software Approach for On-Line Soft Error Mitigation in Interrupt-Driven Applications

Integrity assurance of configuration data has a significant impact on microcontroller-based systems reliability. This is especially true when running applications driven by events which behavior is tightly coupled to this kind of data. This work proposes a new hybrid technique that combines hardware and software resources for detecting and recovering soft-errors in system configuration data. Our approach is based on the utilization of a common built-in microcontroller resource (timer) that works jointly with a software-based technique, which is responsible to periodically refresh the configuration data. The experiments demonstrate that non-destructive single event effects can be effectively mitigated with reduced overheads. Results show an important increase in fault coverage for SEUs and SETs, about one order of magnitude.

Capitalism without Capitalists? A Bottom-Up View of Industrial Transformation in East Germany. CES Germany & Europe Working Papers, No. 00.1, 1999

Research on the industrial transition in East Germany and its outcomes has long focused on the strategy of the Treuhand­ anstalt (IHA). According to institutionalists, David Stark and Lazlo Brust!: (1998), the powerful position of the German privatization agency was not only a result of German unification but also a function of a pathway rooted in the institutional peculiarities of the East German economy before 1989. This paper shows that neither a simple top-down perspective nor the pathway approach, as Stark and Brust!: suggested, are adequate for explaining the internal dynamic of enterprise transformation as well as the outcomes of this process. First of all, the dissolution of the former organizational structures and hierarchies was less coordinated by the 1HA than is often assumed. Often Kombinates fell apart more quickly from below than they could be dismantled from above since enterprises or their units chose the exit­ option and had good reasons to do so. Secondly, although the privatization by the 1HA resulted in the clear dominance of Western investors, the new ownership structure of East German industry as a whole could be characterized as a "capitalism without (East German) capitalists." In fact, what exists in East Germany is rather a kind of "small business capitalism" (KleinbetriebsknpitalifmllS) in which small-and medium-sized producers dominate the landscape. Finally, there was no single starting point in 1989. Two different industrial orders shaped the industrial history of the East German regions which were not destroyed between 1945-89, but rather transformed into the state socialist production system. It can be shown that these older historical patterns are relevant for transition and their outcomes as well.

Sedimentology at the continental margin of the southern Weddell Sea

During four expeditions with RV "Polarstern" at the continental margin of the southern Weddell Sea, profiling and geological sampling were carried out. A detailed bathymetric map was constructed from echo-sounding data. Sub-bottom profiles, classified into nine echotypes, have been mapped and interpreted. Sedimentological analyses were carried out on 32 undisturbed box grab surface samples, as well as on sediment cores from 9 sites. Apart from the description of the sediments and the investigation of sedimentary structures on X-radiographs the following characteristics were determined: grain-size distributions; carbonate and Corg content; component distibutions in different grain-size fractions; stable oxygen and carbon isotopes in planktic and, partly, in benthic foraminifers; and physical properties. The stratigraphy is based On 14C-dating, oxygen isotope Stages and, at one site, On paleomagnetic measurements and 230Th-analyses The sediments represent the period of deposition from the last glacial maximum until recent time. They are composed predominantly of terrigenous components. The formation of the sediments was controlled by glaciological, hydrographical and gravitational processes. Variations in the sea-ice coverage influenced biogenic production. The ice sheet and icebergs were important media for sediment transport; their grounding caused compaction and erosion of glacial marine sediments on the outer continental shelf. The circulation and the physical and chemical properties of the water masses controlled the transport of fine-grained material, biogenic production and its preservation. Gravitational transport processes were the inain mode of sediment movements on the continental slope. The continental ice sheet advanced to the shelf edge and grounded On the sea-floor, presumably later than 31,000 y.B.P. This ice movement was linked with erosion of shelf sediments and a very high sediment supply to the upper continental slope from the adiacent southern shelf. The erosional surface On the shelf is documented in the sub-bottom profiles as a regular, acoustically hard reflector. Dense sea-ice coverage above the lower and middle continental slope resulted in the almost total breakdown of biogenic production. Immediately in front of the ice sheet, above the upper continental slope, a <50 km broad coastal polynya existed at least periodically. Biogenic production was much higher in this polynya than elsewhere. Intense sea-ice formation in the polynya probably led to the development of a high salinity and, consequently, dense water mass, which flowed as a stream near bottom across the continental slope into the deep sea, possibly contributing to bottom water formation. The current velocities of this water mass presumably had seasonal variations. The near-bottom flow of the dense water mass, in combination with the gravity transport processes that arose from the high rates of sediment accumulation, probably led to erosion that progressed laterally from east to West along a SW to NE-trending, 200 to 400 m high morphological step at the continental slope. During the period 14,000 to 13,000 y.B.P., during the postglacial temperature and sea-level rise, intense changes in the environmental conditions occured. Primarily, the ice masses on the outer continental shelf started to float. Intense calving processes resulted in a rapid retreat of the ice edge to the south. A consequence of this retreat was, that the source area of the ice-rafted debris changed from the adjacent southern shelf to the eastern Weddell Sea. As the ice retreated, the gravitational transport processes On the continental slope ceased. Soon after the beginning of the ice retreat, the sea-ice coverage in the whole research area decreased. Simultaneously, the formation of the high salinity dense bottom water ceased, and the sediment composition at the continental slope then became influenced by the water masses of the Weddell Gyre. The formation of very cold Ice Shelf Water (ISW) started beneath the southward retreating Filchner-Ronne Ice Shelf somewhat later than 12,000 y.B.P. The ISW streamed primarily with lower velocities than those of today across the continental slope, and was conducted along the erosional step on the slope into the deep sea. At 7,500 y.B.P., the grounding line of the ice masses had retreated > 400 km to the south. A progressive retreat by additional 200 to 300 km probably led to the development of an Open water column beneath the ice south of Berkner Island at about 4,000 y.B.P. This in turn may have led to an additional ISW, which had formed beneath the Ronne Ice Shelf, to flow towards the Filcher Ice Shelf. As a result, increased flow of ISW took place over the continental margin, possibly enabling the ISW to spill over the erosional step On the upper continental slope towards the West. Since that time, there is no longer any documentation of the ISW in the sedimentary Parameters on the lower continental slope. There, recent sediments reflect the lower water masses of the Weddell Gyre. The sea-ice coverage in early Holocene time was again so dense that biogenic production was significantly restricted.

Element analyses and isotopic composition of basalts from the western Pacific

New Sr- Nd- and Pb-isotopic and trace element data are presented on basalts from the Sulu and Celebes Basins, and the submerged Cagayan Ridge Arc (Western Pacific), recently sampled during Ocean Drilling Program Leg 124. Drilling has shown that the Sulu Basin developed about 18 Ma ago as a backarc basin, associated with the now submerged Cagayan Ridge Arc, whereas the Celebes Basin was generated about 43 Ma ago, contemporaneous with a general plate reorganisation in the Western Pacifc, subsequently developing as an open ocean receiving pelagic sediments until the middle Miocene. In both basins, a late middle Miocene collision phase and the onset of volcanic activity on adjacent arcs in the late Miocene are recorded. Covariations between 87Sr/86Sr and 143Nd/144Nd show that the seafoor basalts from both the Sulu and Celebes Basins are isotopically similar to depleted Indian mid-ocean ridge basalts (MORB), and distinct from East Pacifc Rise MORB, defining a single negative correlation. The Cagayan Arc volcanics are different, in that they have distinctly lower epsilon-Ne(T) for a given epsilon-Sr(T), compared to Sulu and Celebes basalts. In the 207Pb/204Pb and 208Pb/204Pb versus 206Pb/204Pb diagrams, the Celebes, Sulu and Cagayan rocks all plot distinctly above the Northern Hemisphere Reference Line, with high Delta 7/4 Pb (5.3-9.3) and Delta 8/4 Pb (46.3-68.1) values. They define a single trend of radiogenic lead enrichment from Celebes through Sulu to Cagayan Ridge, within the Indian Ocean MORB data field. The data suggest that the overall chemical and isotopic features of the Sulu, Cagayan and Celebes rocks may be explained by partial melting of a depleted asthenospheric N-MORB-type ("normal") mantle source with isotopic characteristics similar to those of the Indian Ocean MORB source. This asthenospheric source was slightly heterogeneous, giving rise to the Sr-Nd isotopic differences between the Celebes and Sulu basalts, and the Cagayan Ridge volcanics. In addition, a probably slab-derived component enriched in LILE and LREE is required to generate the elemental characteristics and low Ne(T) of the Cagayan Ridge island arc tholeiitic and calcalkaline lavas, and to contribute to a small extent in the backarc basalts of the Sulu Sea. The results of this study confirm and extend the widespread Indian Ocean MORB signature in the Western Pacifc region. This signature could have been inherited by the Indian Ocean mantle itself during the rupture of Gondwanaland, when fragments of this mantle could have migrated towards the present position of the Celebes, Sulu and Cagayan sources.

Effects of thallus disturbance on abundance and biomass of soft-bottom species in Kongsfjorden in summer 2013

The effects of biotic disturbances, like seaweed whiplash, on the diversity of benthic communities are well documented for temperate coastal systems, yet missing for Arctic benthos. In Arctic soft-bottom habitats, kelp thalli occur either continuously (e.g. trapped by sediment) or sporadically (by drifting on the sediment) after detachment from rocky shores. To explore whether a kelp thallus can disturb the structure and diversity of a coastal Arctic soft-bottom assemblage, we continuously fixed a single thallus of the kelp Saccharina latissima to or sporadically (i.e. biweekly) moved it on the sediment and compared treatment effects to unmanipulated plots (= controls). On 6 September 2013 (i.e. after 73 days of manipulation), one sediment core was taken from each of the 30 plots (n = 10), from which the number of individuals of each of the 45 encountered animal species were recorded. The continuous presence of an experimentally fixed kelp thallus significantly reduced the number of individuals on average by 27 %. This disturbance effect was even stronger, on average 49 %, where a kelp thallus was biweekly moved on the sediment. Likewise, taxon richness was lowered by an average of 19 and 36 % where a S. latissima thallus was continuously or sporadically present, respectively. While the composition of taxa was also significantly different among all treatment groups, evenness and biomass were unaffected by kelp treatments. We conclude that the presence and already movements of a single kelp thallus can promote small scale patchiness in near-shore soft-bottom assemblage structure and diversity and exemplify a significant connection between rocky and sedimentary coastal habitats.

Growth, viral production and metabolism of a Helicoverpa zea cell line in serum-free culture

Insect cell cultures have been extensively utilised for means of production for heterologous proteins and biopesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five(TM)) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines have been investigated thoroughly over recent years. The Helicoverpa zea cell line has potential use for the production of a biopesticide, specifically the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). The growth, virus production, nutrient consumption and waste production of this cell line was investigated under serum-free culture conditions, using SF900II and a low cost medium prototype (LCM). The cell growth ( growth rates and population doubling time) was comparable in SF900II and LCM, however, lower biomass and cell specific virus yields were obtained in LCM. H. zea cells showed a preference for asparagine over glutamine, similar to the High Five(TM) cells. Ammonia was accumulated to significantly high levels (16 mM) in SF900II, which is an asparagine and glutamine rich medium. However, given the absence of asparagine and glutamine in the medium ( LCM), H. zea cells adapted and grew well in the absence of these substrates and no accumulation of ammonia was observed. The adverse effect of ammonia on H. zea cells is unknown since good production of biologically active HaSNPV was achieved in the presence of high ammonia levels. H. zea cells showed a preference for maltose even given an abundance supply of free glucose. Accumulation of lactate was observed in H. zea cell cultures.

Tetracycline-Inducible packaging cell line for production of Flavivirus replicon particles.

We have previously developed replicon vectors derived from the Australian flavivirus Kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. To facilitate further applications of these vectors in the form of virus-like particles (VLPs), we have now generated a stable BHK packaging cell line, tetKUNCprME, carrying a Kunjin structural gene cassette under the control of a tetracycline-inducible promoter. Withdrawal of tetracycline from the medium resulted in production of Kunjin structural proteins that were capable of packaging transfected and self-amplified Kunjin replicon RNA into the secreted VLPs at titers of up to 1.6 x 10(9) VLPs per ml. Furthermore, secreted KUN replicon VLPs from tetKUNCprME cells could be harvested continuously for as long as 10 days after RNA transfection, producing a total yield of more than 1010 VLPs per 106 transfected cells. Passaging of VLPs on Vero cells or intracerebral injection into 2- to 4-day-old suckling mice illustrated the complete absence of any infectious Kunjin virus. tetKUNCprME cells were also capable of packaging replicon RNA from closely and distantly related flaviviruses, West Nile virus and dengue virus type 2, respectively. The utility of high-titer KUN replicon VLPs was demonstrated by showing increasing CD8(+)-T-cell responses to encoded foreign protein with increasing doses of KUN VLPs. A single dose of 2.5 x 10(7) VLPs carrying the human respiratory syncytial virus M2 gene induced 1,400 CD8 T cells per 10(6) splenocytes in an ex vivo gamma interferon enzyme-linked immunospot assay. The packaging cell line thus represents a significant advance in the development of the noncytopathic Kunjin virus replicon-based gene expression system and may be widely applicable to the basic studies of flavivirus RNA packaging and virus assembly as well as to the development of gene expression systems based on replicons from different flaviviruses.