Differentiating embryonal stem cells are a rich source of haemopoietic gene products and suggest erythroid preconditioning of primitive haemopoietic stem cells

The difficulties associated with studying molecular mechanisms important in hemopoietic stem cell (HSC) function such as the problems of purifying homogeneous stem cell populations, have prompted us to adapt the murine ES cell system as an in vitro model of HSC generation and function. We now report that careful analysis of the time course of HSC generation in differentiating ES cells allows them to be used as a source of known and novel hemopoietic gene products. We have generated a subtracted library using cDNA from ES cells collected just prior to and just following the emergence of HSCs. Analysis of this library shows it to be a rich source of known hemopoietic and hemopoietic related gene products with 44% of identifiable cDNAs falling into these camps. We have demonstrated the value of this system as a source of novel genes of relevance to HSC function by characterizing a novel membrane protein encoding cDNA that is preferentially expressed in primitive hemopoietic cells. Intriguingly, further analysis of the known components of the subtracted library is suggestive of erythroid preconditioning of the ES cell-derived HSC. We have used dot-blot and in situ analysis to indicate that this erythroid preconditioning is probably restricted to primitive but not definitive HSC.     

Different domains of the essential GTPase Cdc42p required for growth and development of Saccharomyces cerevisiae

In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth. Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth. In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression. In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth. Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions. Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.     

Single-chain antibody displayed on a recombinant measles virus confers entry through the tumor-associated carcinoembryonic antigen

To redirect the tropism of the vaccine strain of measles virus (MV), Edmonston B, to a targeted cell population, we displayed on the viral hemagglutinin (H) a single-chain antibody (scAb) specific for the tumor-associated carcinoembryonic antigen (CEA). We generated H fusion proteins with three forms of the scAb appended, differing in the lengths of the linkers separating the VH and VL domains and thus in the oligomerization states of the scAbs. All proteins were stable, appeared properly folded, and were transported to the cell surface, but only H displaying the long-linker form of scAb was functional in supporting cell-cell fusion. This protein induced extensive syncytia in cells expressing the normal virus receptor CD46 and also in CD46-negative cells expressing the targeted receptor, human CEA. Replication-competent MV with H replaced by H displaying the long-linker form of scAb was recovered and replicated efficiently in both CD46-positive and CD46-negative, CEA-positive cells. Thus, MV not only tolerates the addition of a scAb on its H protein but also infects cells via a novel interaction between the scAb and its targeted receptor.     

The phosphatidylethanolamine-binding protein is the prototype of a novel family of serine protease inhibitors

Serine proteases are involved in many processes in the nervous system and specific inhibitors tightly control their proteolytic activity. Thrombin is thought to play a role in tissue development and homeostasis. To date, protease nexin-1 is the only known endogenous protease inhibitor that specifically interferes with thrombotic activity and is expressed in the brain. In this study, we report the detection of a novel thrombin inhibitory activity in the brain of protease nexin-1(-/-) mice. Purification and subsequent analysis by tandem mass spectrometry identified this protein as the phosphatidylethanolamine-binding protein (PEBP). We demonstrate that PEBP exerts inhibitory activity against several serine proteases including thrombin, neuropsin, and chymotrypsin, whereas trypsin, tissue type plasminogen activator, and elastase are not affected. Since PEBP does not share significant homology with other serine protease inhibitors, our results define it as the prototype of a novel class of serine protease inhibitors. PEBP immunoreactivity is found on the surface of Rat-1 fibroblast cells and although its sequence contains no secretion signal, PEBP-H(6) can be purified from the conditioned medium upon recombinant expression.     

Levels of linoleic acid and saturated and monounsaturated fatty acids in selected lipid fractions of the serum and platelets in patients with coronary arterial disease

The levels of serum fatty acids in the serum and in the serum fractions of cholesterol esters (ECH) and triglycerides (TG) and the levels of these acids in these fractions of platelets were compared in healthy controls and in patients with clinically manifested coronary arterial disease. Decreased level of linoleic acid was found in the serum and in the ECH fraction of the serum in the patients, with a rise in the level of palmitic acid in the ECH fraction of the serum of these patients. The level of linoleic acid in the ECH and TG platelet fractions in these patients was not different from that in the healthy controls, while in the platelet TG fraction of the patients the level of palmitoleic acid was raised, and the level of oleic acid was increased in the platelet ECH fraction.     

Effects of salinity on turgor pressure and fertility in Tolypella (Characeae)

We present the first experimental results on salinity tolerance and regulation mechanisms in the genus Tolypella. The two species investigated, T. nidifica and T. glomerata, regulate turgor pressure with almost complete effectiveness by adjustment of K+ and CT concentrations. Sucrose is also involved. The mechanism is basically identical to the mechanism of turgor pressure regulation previously identified in representatives of the genera Chara and Lamprothamnium. Since Chara and Lamprothamnium on the one hand and Tolypella on the other belong to different phylogenetic branches that separated early in the geological history of the Characeae, the K+ regulation mechanism can be assumed to represent an ancient pattern derived from a salt-tolerant common ancestor. Furthermore, our experiments provide evidence that salinity is a limiting factor for fertility in both T. nidifica and T. glomerata. Although the onset of gametangia covers the whole range of salinities tested here (0–29 psu), 12psu was the inhibitory level for the formation of mature oospores. Fertilization is probably disturbed by an increase in salinity. An inability to reproduce sexually under euryhaline conditions could explain why the distribution of the two species is restricted to oligo- and mesohaline environments, despite the wide range of salinity tolerance of their vegetative apparatus.     

Dispersed oil disrupts microbial pathways in pelagic food webs

Most of the studies of microbial processes in response to the Deepwater Horizon oil spill focused on the deep water plume, and not on the surface communities. The effects of the crude oil and the application of dispersants on the coastal microbial food web in the northern Gulf of Mexico have not been well characterized even though these regions support much of the fisheries production in the Gulf. A mesocosm experiment was carried out to determine how the microbial community off the coast of Alabama may have responded to the influx of surface oil and dispersants. While the addition of glucose or oil alone resulted in an increase in the biomass of ciliates, suggesting transfer of carbon to higher trophic levels was likely; a different effect was seen in the presence of dispersant. The addition of dispersant or dispersed oil resulted in an increase in the biomass of heterotrophic prokaryotes, but a significant inhibition of ciliates, suggesting a reduction in grazing and decrease in transfer of carbon to higher trophic levels. Similar patterns were observed in two separate experiments with different starting nutrient regimes and microbial communities suggesting that the addition of dispersant and dispersed oil to the northern Gulf of Mexico waters in 2010 may have reduced the flow of carbon to higher trophic levels, leading to a decrease in the production of zooplankton and fish on the Alabama shelf.     

Plankton composition and water quality in a pond of spring origin in Turkey

Phyto/zooplankton composition, chlorophyll a, and some water quality parameters were investigated in a spring-originated pond in Central Anatolia between February 2001 and January 2002. Water temperature, pH, dissolved oxygen, Secchi depth, total and calcium hardness, nitrate-nitrogen, nitrite-nitrogen, ammonia-nitrogen, total phosphorus, and soluble reactive phosphorus levels were analyzed. A total of 49 species belonging to Bacillariophyceae, Chlorophyceae, Cyanophyceae, Cryptophyceae, and Dinophyceae were identified. The highest phytoplankton abundance was found in August, whereas the lowest was determined in January. Phytoplankton abundance increased from February to August and declined in the following months. The Bacillariophyceae were dominant in the phytoplankton community. A total of 21 species of Rotifera, 2 species of Cladocera, and 1 genus of Copepoda were found. The zooplankton community was dominated by Rotifera. The highest abundance of zooplankton was recorded in July and the lowest value in November. The annual mean concentration of chlorophyll a was measured as 1.90 μg l⁻¹. In spite of these eutrophic levels (mean values of total phosphorus and nitrate-nitrogen: 0.069 mg P l⁻¹ and 0.68 mg N l⁻¹), phytoplankton cannot grow satisfactorily because of the short water retention time (0.6 day⁻¹). The shallowness of the pond together with the low phytoplankton biomass and the high concentrations of nutrients are discussed.     

Calf thymus RF-C as an essential component for DNA polymerase delta and epsilon holoenzymes function.

By using a complementation assay that enabled DNA polymerase delta and DNA polymerase epsilon to replicate a singly-DNA primed M13 DNA in the presence of proliferating cell nuclear antigen (PCNA) and Escherichia coli single-stranded DNA binding protein (SSB), we have purified from calf thymus in a five step procedure a multipolypeptide complex with molecular masses of polypeptides of 155, 70, 60, 58, 39 (doublet), 38 (doublet) and 36 kDa. The protein is very likely replication factor C (Tsurimoto, T. and Stillman, B. (1989) Mol. Cell. Biol. 9, 609-619). This conclusion is based on biochemical and physicochemical data and the finding that it contains a DNA stimulated ATPase which is under certain conditions stimulated by PCNA. Together RF-C, PCNA and ATP convert DNA polymerases delta and epsilon to holoenzyme forms, which were able to replicate efficiently SSB-covered singly-DNA primed M13 DNA. Calf thymus RF-C could form a primer recognition complex on a 3'-OH primer terminus in the presence of calf thymus PCNA and ATP. Holoenzyme complexes of DNA polymerase delta and epsilon could be isolated suggesting that these enzymes directly interact with the auxiliary proteins in a similar way. Under optimal replication conditions on singly-DNA primed M13 DNA the DNA synthesis rate of DNA polymerase delta was higher than of DNA polymerase epsilon. Based on these functional date possible roles of these two DNA polymerases in eukaryotic DNA replication are discussed.     

A flagellum-specific calcium sensor

The flagellar calcium-binding protein (FCaBP) of the flagellated protozoan Trypanosoma cruzi associates with the flagellar membrane via its N-terminal myristate and palmitate moieties in a calcium-modulated, conformation-dependent manner. This mechanism of localization is similar to that described for neuronal calcium sensors, which undergo calcium-dependent changes in conformation, which modulate the availability of the acyl groups for membrane interaction and partner association. To test whether FCaBP undergoes a calcium-dependent conformational change and to explore the role of such a change in flagellar targeting, we first introduced point mutations into each of the two EF-hand calcium-binding sites of FCaBP to define their affinities. Analysis of recombinant EF-3 mutant (E151Q), EF-4 mutant (E188Q), and double mutant proteins showed EF-3 to be the high affinity site (Kd approximately 9 microM) and EF-4 the low affinity site (Kd approximately 120 microM). These assignments also correlated with partial (E188Q), nearly complete (E151Q), and complete (E151Q,E188Q) disruption of calcium-induced conformational changes determined by NMR spectrometry. We next expressed the FCaBP E151Q mutant and the double mutant in T. cruzi epimastigotes. These transproteins localized to the flagellum, suggesting the existence of a calcium-dependent interaction of FCaBP that is independent of its intrinsic calcium binding capacity. Several proteins were identified by FCaBP affinity chromatography that interact with FCaBP in a calcium-dependent manner, but with differential dependence on calcium-binding by FCaBP. These findings may have broader implications for the calcium acyl switch mechanism of protein regulation.