Effect of suppression of erythropoiesis on hematopoietic stem cells in the mouse

Erythropoiesis was suppressed in CDF₁ female mice using one of three different techniques: hypertransfusion, daily injections of low doses of actinomycin D, or post-hypobaric polycythemia. Spleens and bone marrows of these mice contained increased numbers of stem cells as determined by transplantation into lethally irradiated syngeneic mice. These stem cells were capable of producing both erythrocytic and granulocytic progeny. An increase in the number of multipotential stem cells or increases in the numbers of both unipotential granulocytic and erythrocytic stem cells is compatible with these observations and implies that hematopoietic stem cells may have a basic rate of division that is in part independent of stimuli to differentiate. The implications of these findings for the possible modification of antineoplastic drug-induced bone marrow toxicity are discussed.     

Characterization of testudine melanomacrophage linear,membrane extension processes—Cablepodia—By phase and atomic force microscopy

Summary Melanomacrophages (MMs) are a component of an internal, pigmented cell system in liver and splenic tissues of some fishes, anurans, and reptiles. The cells have been found in centers or aggregates in sinusoids and are associated with cells capable of producing a peptide cytokine and immunoglobulins. A unique cell extension process has been observed in turtle MMs placed into cell culture, and this process has been studied by light and atomic force microscopy. These structures, referred to as cablepodia, are uniquely straight, narrow, and unbranching and appear to originate from growth cones opposite lamellipodia. Cablepodia were found to connect with other turtle MMs and fibroblasts forming cell networks. Dividing fibroblasts to which a cablepodium attached ceased cell division. The observations collectively suggest that a principal reason for aggregations of MMs in internal organs of lower vertebrates in their ability to form interconnected networks of cell processes for trapping and processing of particulate matter, cells and infectious organisms and, possibly, for the communication of cell signals and transfer of intracellular materials.     

Regulation of β-catenin trafficking to the membrane in living cells

β-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3β. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of β-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged β-catenin, and found that a small mobile pool of β-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t_0.5 of ~ 30 s. Thus, β-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of β-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t_0.5 ~5 s) is indicative of high turnover and transient association. In contrast, β-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t_0.5 ~8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of β-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than β-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3β increased β-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of β-catenin. In summary, β-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis.     

Contraceptive potential of antibodies to the zona pellucida

The notion of a contraceptive vaccine based on gamete-specific surface antigens was first proposed over a decade ago, as the result of in-vitro and in-vivo studies, and in recent years has been the subject of intensive research. In particular, the zona pellucida has attracted much attention as a potential target for immunological intervention in the fertilization process. Such is the rapidly-expanding nature of research into the biochemical and biological characterization of this structure, that a review of the implications for the development of a contraceptive vaccine seems timely.     

Purification, sequencing and functions of calreticulin from maize

The most abundant proteins in the lumen of the endoplasmic reticulum(ER) are thought to be molecular chaperones, some of which mightalso be involved in calcium storage and release. We have purifiedcalreticulin from maize by ion exchange and reverse-phase chromatography.Identity with plant and animal calreticulins was confirmed byN-terminal amino acid sequencing and it was shown to bind calciumwith a calcium overlay technique. An antiserum raised to thepurified protein was used to screen an expression library andthe full coding sequence for maize calreticulin was determinedfrom the clones selected. The sequence shows 96% identity tobarley calreticulin and 55% identity to animal calreticulins.The three major functional regions are conserved, as are targetingand retention features. When visualized by indirect immunofluorescencemicroscopy, calreticulin was found to be confined to the ERand nuclear envelope of maize root cells. It was distributedthroughout the ER compartment and we found no evidence of calreticulin-enriched areas of ER, such as might be associated with specializedcalcium storage domains. Increasing or decreasing extracellularcalcium did not induce measurable changes in calreticulin levels.In addition, maize calreticulin, as well as other recognizedchaperones, was shown to bind to denatured protein and couldbe eluted specifically by nucleoside trisphosphates. Key words: Endoplasmic reticulum, calcium-binding protein, immunofluorescence, targeting, Zea mays L     

Blockade of human group X secreted phospholipase A2 (GX-sPLA2)-induced airway inflammation and hyperresponsiveness in a mouse asthma model by a selective GX-sPLA2 inhibitor

Group X (GX) phospholipase A(2), a member of a large group of secreted phospholipases A(2) (sPLA(2)s), has recently been demonstrated to play an important in vivo role in the release of arachidonic acid and subsequent formation of eicosanoids. In a Th2 cytokine-driven mouse asthma model, deficiency of mouse GX (mGX)-sPLA(2) significantly impairs development of the asthma phenotype. In this study, we generated mGX-sPLA(2)(-/-) mice with knock-in of human GX (hGX)-sPLA(2) (i.e. hGX-sPLA(2)(+/+) knock-in mice) to understand more fully the role of GX-sPLA(2) in these allergic pulmonary responses and to assess the effect of pharmacological blockade of the GX-sPLA(2)-mediated responses. Knock-in of hGX-sPLA(2) in mGX-sPLA(2)(-/-) mice restored the allergen-induced airway infiltration by inflammatory cells, including eosinophils, goblet cell metaplasia, and hyperresponsiveness to methacholine in the mGX-sPLA(2)-deficient mice. This knock-in mouse model enabled the use of a highly potent indole-based inhibitor of hGX-sPLA(2), RO061606 (which is ineffective against mGX-sPLA(2)), to assess the potential utility of GX-sPLA(2) blockade as a therapeutic intervention in asthma. Delivery of RO061606 via mini-osmotic pumps enabled the maintenance in vivo in the mouse asthma model of plasma inhibitor concentrations near 10 μm, markedly higher than the IC(50) for inhibition of hGX-sPLA(2) in vitro. RO061606 significantly decreased allergen-induced airway inflammation, mucus hypersecretion, and hyperresponsiveness in the hGX-sPLA(2)(+/+) knock-in mouse. Thus, development of specific hGX-sPLA(2) inhibitors may provide a new pharmacological opportunity for the treatment of patients with asthma.     

Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(H(c)): antigen E) by Pichia pastoris

A process was developed for production of a candidate vaccine antigen, recombinant C-terminal heavy chain fragment of the botulinum neurotoxin serotype E, rBoNTE(H(c)) in Pichia pastoris. P. pastoris strain GS115 was transformed with the rBoNTE(H(c)) gene inserted into pHILD4 Escherichia coli-P. pastoris shuttle plasmid. The clone was characterized for genetic stability, copy number, and BoNTE(H(c)) sequence. Expression of rBoNTE(H(c)) from the Mut(+) HIS4 clone was confirmed in the shake-flask, prior to developing a fed-batch fermentation process at 5 and 19 L scale. The fermentation process consists of a glycerol growth phase in batch and fed-batch mode using a defined medium followed by a glycerol/methanol transition phase for adaptation to growth on methanol and a methanol induction phase resulting in the production of rBoNTE(H(c)). Specific growth rate, ratio of growth to induction phase, and time of induction were critical for optimal rBoNTE(H(c)) production and minimal proteolytic degradation. A computer-controlled exponential growth model was used for process automation and off-gas analysis was used for process monitoring. The optimized process had an induction time of 9 h on methanol and produced up to 3 mg of rBoNTE(H(c)) per gram wet cell mass as determined by HPLC and Western blot analysis.     

Over‐expression of specific HvCslF cellulose synthase‐like genes in transgenic barley increases the levels of cell wall (1,3;1,4)‐β‐d‐glucans and alters their fine structure

Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)‐β‐d ‐glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)‐β‐d ‐glucans in the Poaceae is mediated, in part at least, by the cellulose synthase‐like CslF family of genes. Over‐expression of the barley CslF6 gene under the control of an endosperm‐specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)‐β‐d ‐glucan content in grain of transgenic barley. Analyses of (1,3;1,4)‐β‐d ‐glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)‐β‐d ‐glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)‐β‐d ‐glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)‐β‐d ‐glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)‐β‐d ‐glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)‐β‐d ‐glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)‐β‐d ‐glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.     

The measurement of chemically-induced DNA repair synthesis in human cells by BND-cellulose chromatography.

Repair synthesis in human cells in tissue culture can be readily separated from semi-conservative DNA synthesis with the aid of a benzoylated naphthoylated DEAE cellulose (BND-cellulose) column. Cells are incubated with a radioactive DNA precursor during treatment with a repair-inducing agent. An inhibitor of semi-conservative DNA synthesis (hydroxyurea) is added to slow the progression of the DNA growing point. The cells are lysed and after treatment with ribonuclease and pronase the lysates are sheared and passed through a BND-cellulose column. Native DNA is eluted with I M NaCl. Any increase in radioactivity in the native DNA is due to repair synthesis and the specific repair activity (nucleotides inserted per mug of DNA) can be determined from radioactivity and absorbancy measurements. Repair can also be measured in the region of the DNA growing point by fractionation of the material eluted from BND-cellulose with 50% formamide. Repair was not detected in N-acetoxy-2-acetylaminofluorene (AAAF)-treated lymphoblasts derived from an individual with xeroderma pigmentosum although methyl methanesulfonate (MMS)-induced repair was observed in these cells.