Directionality of heart looping: effects of Pitx2c misexpression on flectin asymmetry and midline structures

The insulin-like growth factors (IGFs) are well known mitogens, both in vivo and in vitro, while functions in cellular differentiation have also been indicated. Here, we demonstrate a new role for the IGF pathway in regulating head formation in Xenopus embryos. Both IGF-1 and IGF-2, along with their receptor IGF-1R, are expressed early during embryogenesis, and the IGF-1R is present particularly in anterior and dorsal structures. Overexpression of IGF-1 leads to anterior expansion of head neural tissue as well as formation of ectopic eyes and cement gland, while IGF-1 receptor depletion using antisense morpholino oligonucleotides drastically reduces head structures. Furthermore, we demonstrate that IGF signaling exerts this effect by antagonizing the activity of the Wnt signal transduction pathway in the early embryo, at the level of beta-catenin. Thus, the IGF pathway is required for head formation during embryogenesis.     

Design, synthesis and properties of novel powerful antioxidants, glutathione analogues

Glutathione (GSH) is the major low-molecular weight antioxidant in mammalian cells. Thus, its analogues carrying similar and/or additional positive properties might have clinical perspectives. Here, we report the design and synthesis of a library of tetrapeptidic GSH analogues called UPF peptides. Compared to cellular GSH our designed peptidic analogues showed remarkably higher hydroxyl radical scavenging ability (EC(50) of GSH: 1,231.0 +/- 311.8 microM; EC(50) of UPF peptides: from 0.03 to 35 microM) and improved antiradical efficiency towards a stable alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical. The best of UPF peptides was 370-fold effective hydroxyl radical scavengers than melatonin (EC(50): 11.4 +/- 1.0 microM). We also found that UPF peptides do not influence the viability and membrane integrity of K562 human erythroleukemia cells even at 200 microM concentration. Dimerization of GSH and UPF peptides was compared in water and in 0.9% saline solutions. The results, together with an earlier finding that UPF1 showed protective effects in global cerebral ischemia model in rats, suggest that UPF peptides might serve both as potent antioxidants as well as leads for design of powerful non-peptidic antioxidants that correct oxidative stress-driven events.     

A microcalorimetric perfusion vessel used for measurements of total activity in sediment samples

A multichannel microcalorimeter of the heat-conduction type was equipped with a prototype of a perfusion vessel and was used to measure the total activity during long time periods in natural, aquatic sedimnet samples from aerobic environments. The effect of addition of organic substances, a pollutant and bacteria was studied. The water phase of the sediment phase of the sediment samples was aerated with humidified air and stirred uring measurements. The total activity of the actual sediment was also measured in an ampoule system, for comparison. Aeration of the samples led to an increased heat production rate.     

AKINETE PRODUCTION OF ANABAENA LEMMERMANNII AND A. CYLINDRICA (CYANOPHYCEAE) IN NATURAL POPULATIONS OF N‐ AND P‐LIMITED COASTAL MESOCOSMS1

A strong biomass increase of two Anabaena species was observed in natural plankton community enclosed into nine large mesocosms (51 m³) and manipulated with mineral nutrients and an organic carbon source during a 3‐week period in the coastal Baltic Sea. The water column and settled material from the bottom of the mesocosms were sampled at 2‐day intervals. Planktonic populations of Anabaena lemmermannii Richter and A. cylindrica Lemmermann and sedimentation rates of akinetes to the bottom were quantified. Comparing mesocosms with artificially induced nitrogen and phosphorus limitation, we found that during the third week of the experiment, the population size of A. lemmermannii was clearly higher in nitrogen‐limited units (by a factor of 2.4), whereas the production rate of akinetes was higher in the phosphorus‐limited units (by a factor of 2.5). Input of freshly produced A. lemmermannii akinetes to the benthos was on average 15 × 10⁶ and 6 × 10⁶ cells· m^?2·d^?1 in the P^? and N^? limited mesocosms, respectively. Our estimates of specific akinete production rate of A. lemmermannii in P^? and N^? limited mesocosms revealed an even larger divergence (a factor of 5.5), being on average 2.4 and 0.4 akinetes·10^?3 vegetative cells^?1·d^?1, respectively. The phosphorus addition effectively reduced akinete production of A. lemmermannii. Differences in the nutrient manipulation had no apparent effect on the biomass and akinete production of A. cylindrica. The akinete production pattern of A. cylindrica revealed a 1‐week delay compared with the vegetative population peak, whereas such a delay was not obvious in A. lemmermannii.     

Direct differential counting compared with testing of isolates as means of assessing hydrolytic capacities of natural microbial populations

The abundance of microorganisms with characteristic, physiological activities in aquatic sediment communities can be estimated either by direct differential counting on selective media or by making isolations followed by testing of the isolates. The two methods have been compared with regard to microorganisms performing hydrolyses of different compounds. The first method was used to examine the effect of a model ecocontaminant on the hydrolytic activities of the sediment population.     

Species pools,community completeness and invasion: disentangling diversity effects on the establishment of native and alien species

Invasion should decline with species richness, yet the relationship is inconsistent. Species richness, however, is a product of species pool size and biotic filtering. Invasion may increase with richness if large species pools represent weaker environmental filters. Measuring species pool size and the proportion realised locally (completeness) may clarify diversity‐invasion relationships by separating environmental and biotic effects, especially if species’ life‐history stage and origin are accounted for. To test these relationships, we added seeds and transplants of 15 native and alien species into 29 grasslands. Species pool size and completeness explained more variation in invasion than richness alone. Although results varied between native and alien species, seed establishment and biotic resistance to transplants increased with species pool size, whereas transplant growth and biotic resistance to seeds increased with completeness. Consequently, species pools and completeness represent multiple independent processes affecting invasion; accounting for these processes improves our understanding of invasion.     

Pollen,diatom and plant macrofossil assemblages indicate a low water level phase of Lake Peipsi at the beginning of the Holocene

During the Fennoscandian ice recession from the eastern Baltic area, the water level in the Lake Peipsi basin was decreasing and reached a minimum at the end of the Younger Dryas Chronozone. The low lake level episode is represented in the basal deposits by a ca. 0.8 m thick bed of coarse detritus gyttja dated to 9.6–9.1 ka ¹⁴C BP. The gyttja lies at an elevation of 20–21 m a.s.l., i.e. about 9 m below the present lake level (30 m a.s.l.), and is buried under a 1.5–2 m bed of fine to medium-grained sand. The overall pollen data indicate a shallow-water littoral environment during the gyttja accumulation in the area studied. The diatom flora in the gyttja is dominated by shallow-water benthic and epiphytic taxa, indicating eutrophic littoral conditions at the time of gyttja accumulation. In the uppermost portion of the gyttja sequence the pollen and diatom successions indicate a short period of land emergence when swampy conditions prevailed in the surroundings.     

Identification of a Receptor for Neuropeptide VGF and Its Role in Neuropathic Pain

VGF (nonacronymic) is a neuropeptide precursor that plays multiple roles in regulation of energy balance, reproduction, hippocampal synaptic plasticity, and pain. Data from a number of pain models showed significant up-regulation of VGF in sensory neurons. TLQP-21, one of the VGF-derived neuropeptides, has been shown to induce a hyperalgesic response when injected subcutaneously into the hind paw of mice. However, the precise role of VGF-derived neuropeptides in neuropathic pain and the molecular identity of the receptor for VGF-derived peptides are yet to be investigated. Here we identified gC1qR, the globular heads of the C1q receptor, as the receptor for TLQP-21 using chemical cross-linking combined with mass spectrometry analysis. TLQP-21 caused an increase in intracellular Ca²⁺ levels in rat macrophages and microglia. Inoculation of TLQP-21-stimulated macrophages into rat hind paw caused mechanical hypersensitivity. The increase in intracellular Ca²⁺ levels in macrophages was attenuated by either siRNA or neutralizing antibodies against gC1qR. Furthermore, application of the gC1qR-neutralizing antibody to rats with partial sciatic nerve ligation resulted in a delayed onset of nerve injury-associated mechanical hypersensitivity. These results indicate that gC1qR is the receptor for TLQP-21 and plays an important role in chronic pain through activation of macrophages. Because direct association between TLQP-21 and gC1qR is required for activation of macrophages and causes hypersensitivity, disrupting this interaction may be a useful new approach to develop novel analgesics.     

Novel anti-apoptotic effect of the retinoblastoma protein: implications for polyamine analogue toxicity

The retinoblastoma protein (pRb) pathway is frequently altered in breast cancer cells. pRb is involved in the regulation of cell proliferation and cell death. The breast cancer cell line L56Br-C1 does not express pRb and is extremely sensitive to treatment with the polyamine analogue N ¹,N ¹¹-diethylnorspermine (DENSPM) which causes apoptosis. Polyamines are essential for the regulation of cell proliferation, cell differentiation and cell death. DENSPM depletes cells of polyamines, e.g., by inducing the activity of the polyamine catabolic enzyme spermidine/spermine N ¹-acetyltransferase (SSAT). In this study, L56Br-C1 cells were transfected with human pRb–cDNA. Overexpression of pRb inhibited DENSPM-induced cell death and DENSPM-induced SSAT activity. This suggests that the pRb protein level is a promising marker for polyamine depletion sensitivity and that there is a connection between pRb and the regulation of SSAT activity. We also show that SSAT protein levels and SSAT activity do not always correlate, suggesting that there is an unknown regulation of SSAT.     

The establishment of 20 different human embryonic stem cell lines and subclones; a report on derivation, culture, characterisation and banking

This report summarises our efforts in deriving, characterising and banking of 20 different human embryonic stem cell lines. We have derived a large number of human embryonic stem cell lines between 2001 and 2005. One of these cell lines was established under totally xeno-free culture conditions. In addition, several subclones have been established, including a karyoptypical normal clone from a trisomic mother line. A master cell banking system has been utilised in concert with an extensive characterisation programme, ensuring a supply of high quality pluripotent stem cells for further research and development. In this report we also present the first data on a proprietary novel antibody, hES-Cellect, that exhibits high specificity for undifferentiated hES cells. In addition to the traditional manual dissection approach of propagating hES cells, we here also report on the successful approaches of feeder-free cultures as well as single cell cultures based on enzymatic digestion. All culture systems used as reported here have maintained the hES cells in a karyotypical normal and pluripotent state. These systems also have the advantage of being the principal springboards for further scale up of cultures for industrial or clinical applications that would require vastly more cells that can be produced by mechanical means.