A genetic determinant that specifically regulates the frequency of hematopoietic stem cells.

The regulation of hematopoietic stem cell (HSC) homeostasis is not well understood. We screened for genetic polymorphisms that were linked to differences between mouse strains in the numbers of long-term reconstituting HSCs or restricted progenitors in the bone marrow. AKR/J mice had significantly higher frequencies and numbers of both HSCs and restricted progenitors in their bone marrow than C57BL/Ka-Thy-1.1 mice. The C57BL/Ka-Thy-1.1 alleles were partially dominant. A locus on chromosome 17, including the H-2 complex, was significantly linked to the frequency of long-term self-renewing HSCs but showed no evidence of linkage to the frequency of restricted progenitors. Conversely, a chromosome 1 locus exhibited suggestive linkage to restricted progenitor frequencies but was not linked to HSC frequency. This demonstrates that there are distinct genetic determinants of the frequencies of HSCs and restricted progenitors in vivo. The AKR/J chromosome 17 locus was not sufficient to increase HSC frequencies when bred onto a C57BL background. This suggests that to affect HSC frequencies, the product(s) of this locus likely depend on interactions with unlinked modifying loci.     

Divergent effects of BAFF on human memory B cell differentiation into Ig-secreting cells

B cell-activating factor belonging to the TNF family (BAFF) plays a critical role in B cell maturation, yet its precise role in B cell differentiation into Ig-secreting cells (ISCs) remains unclear. In this study, we find that upon isolation human naive and memory B (MB) cells have prebound BAFF on their surface, whereas germinal center (GC) B cells lack detectable levels of prebound BAFF. We attribute their lack of prebound BAFF to cell activation, because we demonstrate that stimulation of naive and MB cells results in the loss of prebound BAFF. Furthermore, the absence of prebound BAFF on GC B cells is not related to a lack of BAFF-binding receptors or an inability to bind exogenous BAFF. Instead, our data suggest that accessibility to soluble BAFF is limited within GCs, perhaps to prevent skewing of the conventional B cell differentiation program. In support of this concept, whereas BAFF significantly enhances ISC differentiation in response to T cell-dependent activation, we report for the first time the ability of BAFF to considerably attenuate ISC differentiation of MB cells in response to CpG stimulation, a form of T cell-independent activation. Our data suggest that BAFF may be providing regulatory signals during specific T cell-independent events, which protect the balance between MB cells and ISCs outside GCs. Taken together, these data define a complex role for BAFF in humoral immune responses and show for the first time that BAFF can also play an inhibitory role in B cell differentiation.     

Lactobacillus casei, Lactobacillus rhamnosus, and Lactobacillus zeae isolates identified by sequence signature and immunoblot phenotype

Species taxonomy within the Lactobacillus casei group of bacteria has been unsettled. With the goal of helping clarify the taxonomy of these bacteria, we investigated the first 3 variable regions of the 16S rRNA gene, the 16S-23S rRNA interspacer region, and one third of the chaperonin 60 gene for Lactobacillus isolates originally designated as L. casei, L. paracasei, L. rhamnosus, and L. zeae. For each genetic region, a phylogenetic tree was created and signature sequence analysis was done. As well, phenotypic analysis of the various strains was performed by immunoblotting. Both sequence signature analysis and immunoblotting gave immediate identification of L. casei, L. rhamnosus, and L. zeae isolates. These results corroborate and extend previous findings concerning these lactobacilli; therefore, we strongly endorse recent proposals for revised nomenclature. Specifically, isolate ATCC 393 is appropriately rejected as the L. casei type strain because of grouping with isolates identified as L. zeae. As well, because all other L. casei isolates, including the proposed neotype isolate ATCC 334, grouped together with isolates designated L. paracasei, we support the use of the single species L. casei and rejection of the name L. paracasei.     

Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material

A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pm sensitivity each consuming only 1 μl of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.     

Photoaffinity labeling of the insulin receptor in H4 hepatoma cells: Lack of cellular receptor processing

Photoaffinity labeling techniques were used to identify insulin-binding components of the plasma membrane in insulin-responsive, monolayer-cultured hepatoma cells. The activated, photosensitive reagent, an n-hydroxysuccinimide ester of 4-azidobenzoic acid, was coupled with highly purifed insulin, and the hormone derivative was subsequently iodinated, bound to cell surface receptors of intact H4 cells, and photoactivatcd. After dissolution of the cells, labeled proteins were analyzed by SDS/polyacrylamide gel electrophoresis under reducing conditions. The main labeled band exhibited an apparent molecular weight of 130,000. Two minor components of apparent mol wt 95,000 and 40,000 were also identified. Specific labeling of all 3 bands was inhibited by simultaneous incubation of the cells with native insulin, but not by the heterologous hormone, glucagon, prior to photoactivation. Binding of azidobenzoyl-insulin to H4 cells was time-dependent, as was the correlated labeling of receptor components. Band-labeling by the photosensitive insulin derivative was totally light-dependent; spontaneous covalent linking of insulin and receptor was not observed. The labeled receptor-related proteins were not degraded by the cells under our experimental conditions.     

Genetic Dissection of Peroxisome-Associated Matrix Protein Degradation in Arabidopsis thaliana

Peroxisomes are organelles that sequester certain metabolic pathways; many of these pathways generate H₂O₂, which can damage proteins. However, little is known about how damaged or obsolete peroxisomal proteins are degraded. We exploit developmentally timed peroxisomal content remodeling in Arabidopsis thaliana to elucidate peroxisome-associated protein degradation. Isocitrate lyase (ICL) is a peroxisomal glyoxylate cycle enzyme necessary for early seedling development. A few days after germination, photosynthesis begins and ICL is degraded. We previously found that ICL is stabilized when a peroxisome-associated ubiquitin-conjugating enzyme and its membrane anchor are both mutated, suggesting that matrix proteins might exit the peroxisome for ubiquitin-dependent cytosolic degradation. To identify additional components needed for peroxisome-associated matrix protein degradation, we mutagenized a line expressing GFP–ICL, which is degraded similarly to endogenous ICL, and identified persistent GFP-ICL fluorescence (pfl) mutants. We found three pfl mutants that were defective in PEROXIN14 (PEX14/At5g62810), which encodes a peroxisomal membrane protein that assists in importing proteins into the peroxisome matrix, indicating that proteins must enter the peroxisome for efficient degradation. One pfl mutant was missing the peroxisomal 3-ketoacyl-CoA thiolase encoded by the PEROXISOME DEFECTIVE1 (PED1/At2g33150) gene, suggesting that peroxisomal metabolism influences the rate of matrix protein degradation. Finally, one pfl mutant that displayed normal matrix protein import carried a novel lesion in PEROXIN6 (PEX6/At1g03000), which encodes a peroxisome-tethered ATPase that is involved in recycling matrix protein receptors back to the cytosol. The isolation of pex6-2 as a pfl mutant supports the hypothesis that matrix proteins can exit the peroxisome for cytosolic degradation.     

Technical report: exploring the basis of congenital myasthenic syndromes in an undergraduate course, using the model organism, Caenorhabditis elegans

Mutations affecting acetylcholine receptors have been causally linked to the development of congenital myasthenic syndromes (CMS) in humans resulting from neuromuscular transmission defects. In an undergraduate Molecular Neurobiology course, the molecular basis of CMS was explored through study of a Caenorhabditis elegans model of the disease. The nicotinic acetylcholine receptor (nAChR), located on the postsynaptic muscle cell membrane, contains a pentameric ring structure comprised of five homologous subunits. In the nematode C. elegans, unc-63 encodes an α subunit of nAChR. UNC-63 is required for the function of nAChR at the neuromuscular junction. Mutations in unc-63 result in defects in locomotion and egg-laying and may be used as models for CMS. Here, we describe the responses of four unc-63 mutants to the cholinesterase inhibitor pyridostigmine bromide (range 0.9–15.6 mM in this study), a treatment for CMS that mitigates deficiencies in cholinergic transmission by elevating synaptic ACh levels. Our results show that 15.6 mM pyridostigmine bromide enhanced mobility in two alleles, depressed mobility in one allele and in N2, while having no effect on the fourth allele. This indicates that while pyridostigmine bromide may be effective at ameliorating symptoms of CMS in certain cases, it may not be a suitable treatment for all individuals due to the diverse etiology of this disease. Students in the Molecular Neurobiology course enhanced their experience in scientific research by conducting an experiment designed to increase understanding of genetic defects of neurological function.     

Pleiotropic Actions of Forskolin Result in Phosphatidylserine Exposure in Primary Trophoblasts

Forskolin is an extract of the Coleus forskholii plant that is widely used in cell physiology to raise intracellular cAMP levels. In the field of trophoblast biology, forskolin is one of the primary treatments used to induce trophoblastic cellular fusion. The syncytiotrophoblast (ST) is a continuous multinucleated cell in the human placenta that separates maternal from fetal circulations and can only expand by fusion with its stem cell, the cytotrophoblast (CT). Functional investigation of any aspect of ST physiology requires in vitro differentiation of CT and de novo ST formation, thus selecting the most appropriate differentiation agent for the hypothesis being investigated is necessary as well as addressing potential off-target effects. Previous studies, using forskolin to induce fusion in trophoblastic cell lines, identified phosphatidylserine (PS) externalization to be essential for trophoblast fusion and showed that widespread PS externalization is present even after fusion has been achieved. PS is a membrane phospholipid that is primarily localized to the inner-membrane leaflet. Externalization of PS is a hallmark of early apoptosis and is involved in cellular fusion of myocytes and macrophages. We were interested to examine whether PS externalization was also involved in primary trophoblast fusion. We show widespread PS externalization occurs after 72 hours when fusion was stimulated with forskolin, but not when stimulated with the cell permeant cAMP analog Br-cAMP. Using a forskolin analog, 1,9-dideoxyforskolin, which stimulates membrane transporters but not adenylate cyclase, we found that widespread PS externalization required both increased intracellular cAMP levels and stimulation of membrane transporters. Treatment of primary trophoblasts with Br-cAMP alone did not result in widespread PS externalization despite high levels of cellular fusion. Thus, we concluded that widespread PS externalization is independent of trophoblast fusion and, importantly, provide evidence that the common differentiation agent forskolin has previously unappreciated pleiotropic effects on trophoblastic cells.