Glucose Metabolism,Islet Architecture,and Genetic Homogeneity in Imprinting of [Ca2+]i and Insulin Rhythms in Mouse Islets

We reported previously that islets isolated from individual, outbred Swiss-Webster mice displayed oscillations in intracellular calcium ([Ca²⁺]_i) that varied little between islets of a single mouse but considerably between mice, a phenomenon we termed “islet imprinting.” We have now confirmed and extended these findings in several respects. First, imprinting occurs in both inbred (C57BL/6J) as well as outbred mouse strains (Swiss-Webster; CD1). Second, imprinting was observed in NAD(P)H oscillations, indicating a metabolic component. Further, short-term exposure to a glucose-free solution, which transiently silenced [Ca²⁺]_i oscillations, reset the oscillatory patterns to a higher frequency. This suggests a key role for glucose metabolism in maintaining imprinting, as transiently suppressing the oscillations with diazoxide, a K_ATP-channel opener that blocks [Ca²⁺]_i influx downstream of glucose metabolism, did not change the imprinted patterns. Third, imprinting was not as readily observed at the level of single beta cells, as the [Ca²⁺]_i oscillations of single cells isolated from imprinted islets exhibited highly variable, and typically slower [Ca²⁺]_i oscillations. Lastly, to test whether the imprinted [Ca²⁺]_i patterns were of functional significance, a novel microchip platform was used to monitor insulin release from multiple islets in real time. Insulin release patterns correlated closely with [Ca²⁺]_i oscillations and showed significant mouse-to-mouse differences, indicating imprinting. These results indicate that islet imprinting is a general feature of islets and is likely to be of physiological significance. While islet imprinting did not depend on the genetic background of the mice, glucose metabolism and intact islet architecture may be important for the imprinting phenomenon.     

Acute biological effects of simulating the whole-body radiation dose distribution from a solar particle event using a porcine model

In a solar particle event (SPE), an unshielded astronaut would receive proton radiation with an energy profile that produces a highly inhomogeneous dose distribution (skin receiving a greater dose than internal organs). The novel concept of using megavoltage electron-beam radiation to more accurately reproduce both the total dose and the dose distribution of SPE protons and make meaningful RBE comparisons between protons and conventional radiation has been described previously. Here, Yucatan minipigs were used to determine the effects of a superficial, SPE-like proton dose distribution using megavoltage electrons. In these experiments, dose-dependent increases in skin pigmentation, ulceration, keratinocyte necrosis and pigment incontinence were observed. Five of 18 animals (one each exposed to 7.5 Gy and 12.5 Gy radiation and three exposed to 25 Gy radiation) developed symptomatic, radiation-associated pneumonopathy approximately 90 days postirradiation. The three animals from the highest dose group showed evidence of mycoplasmal pneumonia along with radiation pneumonitis. Moreover, delayed-type hypersensitivity was found to be altered, suggesting that superficial irradiation of the skin with ionizing radiation might cause immune dysfunction or dysregulation. In conclusion, using total doses, patterns of dose distribution, and dose rates that are compatible with potential astronaut exposure to SPE radiation, animals experienced significant toxicities that were qualitatively different from toxicities previously reported in pigs for homogeneously delivered radiation at similar doses.     

Breeding system evolution influenced the geographic expansion and diversification of the core Corvoidea (Aves: Passeriformes)

Birds vary greatly in their life‐history strategies, including their breeding systems, which range from brood parasitism to a system with multiple nonbreeding helpers at the nest. By far the most common arrangement, however, is where both parents participate in raising the young. The traits associated with parental care have been suggested to affect dispersal propensity and lineage diversification, but to date tests of this potential relationship at broad temporal and spatial scales have been limited. Here, using data from a globally distributed group of corvoid birds in concordance with state‐dependent speciation and extinction models, we suggest that pair breeding is associated with elevated speciation rates. Estimates of transition between breeding systems imply that cooperative lineages frequently evolve biparental care, whereas pair breeders rarely become cooperative. We further highlight that these groups have differences in their spatial distributions, with pair breeders overrepresented on islands, and cooperative breeders mainly found on continents. Finally, we find that speciation rates appear to be significantly higher on islands compared to continents. These results imply that the transition from cooperative breeding to pair breeding was likely a significant contributing factor facilitating dispersal across tropical archipelagos, and subsequent world‐wide phylogenetic expansion among the core Corvoidea.     

Synthesis and evaluation of near-infrared fluorescent sulfonamide derivatives for imaging of hypoxia-induced carbonic anhydrase IX expression in tumors

A series of human carbonic anhydrase (hCA) IX inhibitors conjugated to various near-infrared fluorescent dyes was synthesized with the aim of imaging hypoxia-induced hCA IX expression in tumor cells in vitro, ex vivo and in vivo. The resulting compounds were profiled for inhibition of transmembrane hCA IX showing a range of potencies from 7.5 to 116 nM and up to 50-fold selectivity over the cytosolic form hCA II. Some of the compounds also showed inhibition selectivity for other transmembrane forms hCA XII and XIV as well. Compounds incubated in vitro with HeLa cells cultured under normoxic and hypoxic conditions detected upregulation of hCA IX under hypoxia by fluorescence microscopy. A pilot in vivo study in HT-29 tumor bearing mice showed significant accumulation of a fluorescent acetazolamide derivative in tumor tissue with little accumulation in other tissues. Approximately 10% of injected dose was non-invasively quantified in tumors by fluorescence molecular tomography (FMT), demonstrating the promise of these new compounds for quantitative imaging of hCA IX upregulation in live animals.     

A new gain‐of‐function mouse line to study the role of Wnt3a in development and disease

Wnt/β‐catenin signals are important regulators of embryonic and adult stem cell self‐renewal and differentiation and play causative roles in tumorigenesis. Purified recombinant Wnt3a protein, or Wnt3a‐conditioned culture medium, has been widely used to study canonical Wnt signaling in vitro or ex vivo. To study the role of Wnt3a in embryogenesis and cancer models, we developed a Cre recombinase activatable Rosa26^Wnt3a allele, in which a Wnt3a cDNA was inserted into the Rosa26 locus to allow for conditional, spatiotemporally defined expression of Wnt3a ligand for gain‐of‐function (GOF) studies in mice. To validate this reagent, we ectopically overexpressed Wnt3a in early embryonic progenitors using the T‐Cre transgene. This resulted in up‐regulated expression of a β‐catenin/Tcf‐Lef reporter and of the universal Wnt/β‐catenin pathway target genes, Axin2 and Sp5. Importantly, T‐Cre; Rosa26^Wnt3a mutants have expanded presomitic mesoderm (PSM) and compromised somitogenesis and closely resemble previously studied T‐Cre; Ctnnb1^ex3 (β‐catenin^GOF) mutants. These data indicate that the exogenously expressed Wnt3a stimulates the Wnt/β‐catenin signaling pathway, as expected. The Rosa26^Wnt3a mouse line should prove to be an invaluable tool to study the function of Wnt3a in vivo.     

Genomic perspective on the photobiology of Halobacterium species NRC-1, a phototrophic,phototactic, and UV-tolerant haloarchaeon

Halobacterium species display a variety of responses to light, including phototrophic growth, phototactic behavior, and photoprotective mechanisms. The complete genome sequence of Halobacterium species NRC-1 (Proc Natl Acad Sci USA 97: 12176–12181, 2000), coupled with the availability of a battery of methods for its analysis makes this an ideal model system for studying photobiology among the archaea. Here, we review: (1) the structure of the 2.57 Mbp Halobacterium NRC-1 genome, including a large chromosome, two minichromosomes, and 91 transposable IS elements; (2) the purple membrane regulon, which programs the accumulation of large quantities of the light-driven proton pump, bacteriorhodopsin, and allows for a period of phototrophic growth; (3) components of the sophisticated pathways for color-sensitive phototaxis; (4) the gas vesicle gene cluster, which codes for cell buoyancy organelles; (5) pathways for the production of carotenoid pigments and retinal, (6) processes for the repair of DNA damage; and (7) putative homologs of circadian rhythm regulators. We conclude with a discussion of the power of systems biology for comprehensive understanding of Halobacterium NRC-1 photobiology. This revised version was published online in June 2006 with corrections to the Cover Date.     

Spatial overlap of a predatory copepod,Acanthocyclops robustus,and its prey in a shallow eutrophic lake

Spatial overlap between Acanthocyclops robustus, with special emphasis on the adult females, and other zooplankton in one basin of a shallow (approximate depth of 2 m) eutrophic lake was studied.Horizontal distribution patterns were analysed on two dates. On both dates, most taxa examined showed large-scale patchiness between the three sections of the lake basin (approximate length of 1.2 km). Similarly, most taxa, with the important exception of the adult female Acanthocyclops robustus, were significantly patchily distributed on the small-scale (i.e. within sections). However, the intensity of such patchiness was, in general, relatively low. There was no consistent evidence of aggregation by the adult females or copepodites and adult males (the latter two were considered together) of the predator in such small-scale prey patches.Diurnal vertical distribution patterns were studied on two 24–25 hour periods. The first period was characterized by calm weather. Adult female, and perhaps male, Acanthocyclops robustus, Chydorus sphaericus, Bosmina Coregoni, Keratella cochlearis, Asplanchna species, Polyarthra vulgaris and Pompholyx sulcata seemed to show diurnal migration patterns, while seven other taxa showed consistent preferences for particular depths. Only copepod nauplii and Daphnia species were approximately evenly distributed. Negative correlations were found between the vertical distributions of the adult female predator and seven of the seventeen potential prey recognized.The first half of the second period was characterised by strong winds which abated during the second half. Most zooplankton taxa showed inconsistent heterogeneous vertical distributions or were homogeneously distributed with vertical heterogeneity developing towards the end of the period. Only Bosmina longirostris and Daphnia species seemed to show vertical migration patterns. Thus, no consistent vertical segregation between predator and prey was detected.     

The brain-derived neurotrophic factor gene confers susceptibility to bipolar disorder: evidence from a family-based association study

Bipolar disorder (BP) is a severe psychiatric disease, with a strong genetic component, that affects 1% of the population worldwide and is characterized by recurrent episodes of mania and depression. Brain-derived neurotrophic factor (BDNF) has been implicated in the pathogenesis of mood disorders, and the aim of the present study was to test for the presence of linkage disequilibrium between two polymorphisms in the BDNF gene and BP in 283 nuclear families. Family-based association test (FBAT) results for the dinucleotide repeat (GT)(N) polymorphism at position -1040 bp showed that allele A3 was preferentially transmitted to the affected individuals (Z=2.035 and P=.042). FBAT results for the val66met SNP showed a significant association for allele G (Z=3.415 and P=.00064). Transmission/disequilibrium test (TDT) haplotype analysis showed a significant result for the 3-G allele combination (P=.000394), suggesting that a DNA variant in the vicinity of the BDNF locus confers susceptibility to BP. Given that there is no direct evidence that either of the polymorphisms we examined alters function, it is unlikely that the actual risk-conferring allele is from these two sites. Rather, the causative site is likely nearby and in linkage disequilibrium with the 3-G haplotype that we have identified.     

Progressive development of the rat osteoblast phenotype in vitro: reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix.

The relationship of cell proliferation to the temporal expression of genes characterizing a developmental sequence associated with bone cell differentiation was examined in primary diploid cultures of fetal calvarial derived osteoblasts by the combined use of autoradiography, histochemistry, biochemistry, and mRNA assays of osteoblast cell growth and phenotypic genes. Modifications in gene expression define a developmental sequence that has 1) three principle periods--proliferation, extracellular matrix maturation, and mineralization--and 2) two restriction points to which the cells can progress but cannot pass without further signals--the first when proliferation is down-regulated and gene expression associated with extracellular matrix maturation is induced, and the second when mineralization occurs. Initially, actively proliferating cells, expressing cell cycle- and cell growth-regulated genes, produce a fibronectin/type I collagen extracellular matrix. A reciprocal and functionally coupled relationship between the decline in proliferative activity and the subsequent induction of genes associated with matrix maturation and mineralization is supported by 1) a temporal sequence of events in which there is an enhanced expression of alkaline phosphatase immediately following the proliferative period, and later, an increased expression of osteocalcin and osteopontin at the onset of mineralization; 2) increased expression of a specific subset of osteoblast phenotype markers, alkaline phosphatase and osteopontin, when proliferation is inhibited by hydroxyurea; and 3) enhanced levels of expression of the osteoblast markers as a function of ascorbic acid-induced collagen deposition, suggesting that the extracellular matrix contributes to both the shutdown of proliferation and the development of the osteoblast phenotype.