Functional characterization of lymphoid cells generated in serum-deprived culture stimulated with stem cell factor and interleukin 7 from normal and autoimmune mice

We have investigated the phenotypic and functional characteristics of murine pre-B cells obtained in semisolid and liquid culture with stem cell factor (SCF) and interleukin 7 (IL-7). Both serum-supplemented and serum-deprived culture conditions were used. The source of bone marrow cells was either normal mice (CD1 and C3H) or the lupus strain of mice MRL/Ipr and its congenic strain MRL/+. SCF (100 ng/ml) and IL-7 (250 ng/ml) supported murine B cell proliferation in vitro from all the murine strains analyzed both in serum-supplemented and serum-deprived conditions. Maximal colony growth was observed in both cases when the factors were used in combination. The growth factors alone induced some colony growth in serum-supplemented cultures but were either ineffective or had modest activity in serum-deprived cultures. Cells harvested from the colonies or generated in liquid cultures and stimulated with SCF + IL-7 in the absence of serum had almost exclusively a pre-B cell phenotype (BP-1+, B220+, slg-, CD4-, CD8-, Mac-1, RB-6-). Both the maximal colony growth in semisolid culture and the maximal number of cells in liquid culture were observed at day 12–14. At this time, the pre-B cells failed to differentiate further and started to die. Pre-B cells generated in vitro were, however, capable of differentiating in vivo. SCID mice injected with 2 × 10⁶ pre-B cells had readily detectable serum levels of IgM (54 ± 26 m?g/ml) and IgG (60 ± 95 m?g/ml) at 4 weeks and 6 weeks posttransplantation, respectively. Mature B and T cells of the donor major histocompatibility complex type were detected in the SCID mice at sacrifice 14 weeks posttransplantation. These data indicate that purified (>80% BP-1+) populations of functional pre-B cells can be grown from murine bone marrow of normal mice as well as of lupus mice in serum-deprived cultures stimulated with SCF and IL-7. These cultures, therefore, provide a highly enriched source of pre-B cells but also contain T cell precursors that differentiate upon adoptive transfer into SCID mice. © 1995 Wiley-Liss, Inc.     

A Novel Short Isoform of Cytosolic PSD-95 Interactor (Cypin) Regulates Neuronal Development

The guanine deaminase cypin (cytosolic PSD-95 interactor) binds to PSD-95 (postsynaptic density protein 95) and regulates dendrite branching by promoting microtubule polymerization. Here, we identify a novel short isoform of cypin, termed cypinS, which is expressed in mouse and human, but not rat, tissues. Cypin and cypinS mRNA and protein levels peak at P7 and P14 in the mouse brain, suggesting a role for these isoforms during development. Interestingly, although cypinS lacks guanine deaminase activity, overexpression of cypinS increases dendrite branching. This increase occurs further away from soma than do increases resulting from overexpression of cypin. In contrast, overexpression of cypin, but not cypinS, decreases dendritic spine density and maturity. This suggests that changes to spines, but not to dendrites, may be dependent on guanine deaminase activity. Furthermore, overexpression of either cypin or cypinS increases miniature excitatory postsynaptic current (mEPSC) frequency, pointing to a presynaptic role for both isoforms. Interestingly, overexpression of cypinS results in a significantly greater increase in frequency than does overexpression of cypin. Thus, cypin and cypinS play distinct roles in neuronal development.     

Inconsistent detection of extinction debts using different methods

The extinction debt, delayed species extinctions following landscape degradation, is a widely discussed concept. But a consensus about the prevalence of extinctions debts is hindered by a multiplicity of methods and a lack of comparisons among habitats. We applied three contrasting species–area relationship methods to test for plant community extinction debts in three habitats which had different degradation histories over the last century: calcareous grassland, heathland and woodland. These methods differ in their data requirements, with the first two using information on past and current habitat area alongside current species richness, whilst the last method also requires data on past species richness. The most data‐intensive, and hence arguably most reliable method, identified extinction debts across all habitats for specialist species, whilst the other methods did not. All methods detected an extinction debt in calcareous grassland, which had undergone the most severe degradation. We conclude that some methods failed to detect an extinction debt, particularly in habitats that have undergone moderate degradation. Data on past species numbers are required for the most reliable method; as such data are rare, extinction debts may be under‐reported.     

Soil carbon saturation: concept,evidence and evaluation

Current estimates of soil C storage potential are based on models or factors that assume linearity between C input levels and C stocks at steady-state, implying that SOC stocks could increase without limit as C input levels increase. However, some soils show little or no increase in steady-state SOC stock with increasing C input levels suggesting that SOC can become saturated with respect to C input. We used long-term field experiment data to assess alternative hypotheses of soil carbon storage by three simple models: a linear model (no saturation), a one-pool whole-soil C saturation model, and a two-pool mixed model with C saturation of a single C pool, but not the whole soil. The one-pool C saturation model best fit the combined data from 14 sites, four individual sites were best-fit with the linear model, and no sites were best fit by the mixed model. These results indicate that existing agricultural field experiments generally have too small a range in C input levels to show saturation behavior, and verify the accepted linear relationship between soil C and C input used to model SOM dynamics. However, all sites combined and the site with the widest range in C input levels were best fit with the C-saturation model. Nevertheless, the same site produced distinct effective stabilization capacity curves rather than an absolute C saturation level. We conclude that the saturation of soil C does occur and therefore the greatest efficiency in soil C sequestration will be in soils further from C saturation.

Involvement of mitogen-activated protein kinases and reactive oxygen species in the inotropic action of ouabain on cardiac myocytes. A potential role for mitochondrial KATP channels

Binding of ouabain to Na⁺/K⁺-ATPase activated multiple signal transduction pathways including stimulation of Src, Ras, p42/44 MAPKs and production of reactive oxygen species (ROS) in rat cardiac myocytes. Inhibition of either Src or Ras ablated ouabain-induced increase in both [Ca²⁺]_i and contractility. While PD98059 abolished the effects of ouabain on [Ca²⁺]_i, it only caused a partial inhibition of ouabain-induced increases in contractility. On the other hand, pre-incubation of myocytes with N-acetyl cysteine (NAC) reduced the effects of ouabain on contractility, but not [Ca²⁺]_i. Furthermore, 5-hydroxydecanoate (5-HD) blocked ouabain-induced ROS production and partially inhibited ouabain-induced increases in contractility in cardiac myocytes. Pre-incubation of myocytes with both 5-HD and PD98059 completely blocked ouabain's effect on contractility. Finally, we found that opening of mitochondrial K_ATP channel by diazoxide increased intracellular ROS and significantly raised contractility in cardiac myocytes. These new findings indicate that ouabain regulates cardiac contractility via both [Ca²⁺]_i and ROS. While activation of MAPKs leads to increases in [Ca²⁺]_i, opening of mitochondrial K_ATP channel relays the ouabain signal to increased ROS production in cardiac myocytes.     

Platelet-derived growth factor D, tissue-specific expression in the eye, and a key role in control of lens epithelial cell proliferation

Platelet-derived growth factor D (PDGF-D), also known as Iris-expressed growth factor, is a member of the PDGF/vascular endothelial growth factor family. The expression of PDGF-D in the eye is tissue-specific. In the anterior segment, it is localized to iris and ciliary body, whereas in the retina, PDGF-D is restricted to the outer plexiform layer. PDGF-D is present in aqueous humor but is not detectable in mature lens or in mouse lens-derived alphaTN4-1 cells. However, it is expressed in rabbit lens-derived N/N1003A cells. N/N1003A cell-conditioned medium stimulates proliferation in rat lens explants, and this is blocked by immunodepletion of PDGF-D. Immunopurified PDGF-D also stimulates cell proliferation in rat lens explants and in NIH 3T3 cells. In organ culture of rat eye anterior segments, anti-PDGF-D strongly inhibits lens epithelial cell proliferation. This finding suggests a major in vivo role for PDGF-D in the mechanisms of coordinated growth of eye tissues. Intervention in the PDGF-D pathway in the eye, perhaps by antibody or blocking peptide, could be useful in the treatment of certain cataracts, including post-operative secondary cataract.     

The high value of logged tropical forests: lessons from northern Borneo

The carbon storage and conservation value of old-growth tropical forests is clear, but the value of logged forest is less certain. Here we analyse >100,000 observations of individuals from 11 taxonomic groups and >2,500 species, covering up to 19?years of post-logging regeneration, and quantify the impacts of logging on carbon storage and biodiversity within lowland dipterocarp forests of Sabah, Borneo. We estimate that forests lost ca. 53% of above-ground biomass as a result of logging but despite this high level of degradation, logged forest retained considerable conservation value: floral species richness was higher in logged forest than in primary forest and whilst faunal species richness was typically lower in logged forest, in most cases the difference between habitats was no greater than ca. 10%. Moreover, in most studies >90% of species recorded in primary forest were also present in logged forest, including species of conservation concern. During recovery, logged forest accumulated carbon at five times the rate of natural forest (1.4 and 0.28?Mg?C?ha^?1?year^?1, respectively). We conclude that allowing the continued regeneration of extensive areas of Borneo??s forest that have already been logged, and are at risk of conversion to other land uses, would provide a significant carbon store that is likely to increase over time. Protecting intact forest is critical for biodiversity conservation and climate change mitigation, but the contribution of logged forest to these twin goals should not be overlooked.     

A physical map of the genomic region on mouse chromosome 3 containing the hindshaker (hsh) mutation

Hindshaker (hsh), a spontaneous, autosomal recessive mouse mutation, displays a developmentally dependent tremor of the hindquarters due to hypomyelination in the CNS. This myelin deficit is followed by progressive, but incomplete, recovery by postnatal day 42. Herein we describe the construction of a genomic contig spanning the interval between the markers D3Mit187 (42.4 cM) and D3Mit232 (45.2 cM) on mouse chromosome 3, which we have previously shown to contain the hsh mutation. A physical map, covering approximately 3.5 Mb, was constructed from a series of overlapping yeast and bacterial artificial chromosomes. A 1.2- to 1.4-Mb segment central to the contig was compared extensively with the syntenic regions in human (chromosome 1q21-q23) and rat (chromosome 2). We present new data on 10 genes erroneously assigned to this area and on another 6 genes previously assigned elsewhere. For absent genes, our work suggests that they are telomeric to the region encompassed in our map. Accordingly, our findings both map the area surrounding the hsh mutation and present important corrections to the current maps in an area rich in genes related to the nervous system.