The CFTR Mutation c.3453G > C (D1152H) Confers an Anion Selectivity Defect in Primary Airway Tissue that Can be Rescued by Ivacaftor

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene variant, c.3453G > C (D1152H), is associated with mild Cystic Fibrosis (CF) disease, though there is considerable clinical variability ranging from no detectable symptoms to lung disease with early acquisition of Pseudomonas aeruginosa. The approval extension of ivacaftor, the first CFTR modulator drug approved, to include D1152H was based on a positive drug response of defective CFTR-D1152H chloride channel function when expressed in FRT cells. Functional analyses of primary human nasal epithelial cells (HNE) from an individual homozygous for D1152H now revealed that while CFTR-D1152H demonstrated normal, wild-type level chloride conductance, its bicarbonate-selective conductance was impaired. Treatment with ivacaftor increased this bicarbonate-selective conductance. Extensive genetic, protein and functional analysis of the nasal cells of this D1152H/D1152H patient revealed a 90% reduction of CFTR transcripts due to the homozygous presence of the 5T polymorphism in the poly-T tract forming a complex allele with D1152H. Thus, we confirm previous observation in patient-derived tissue that 10% normal CFTR transcripts confer normal, wild-type level chloride channel activity. Together, this study highlights the benefit of patient-derived tissues to study the functional expression and pharmacological modulation of CF-causing mutations, in order to understand pathogenesis and therapeutic responses.

Expression of C/EBPβ in Bone Marrow Mesenchymal Stem Cells Is Mandatory for Early-Stage B Cell Lymphopoiesis

Abstract 3457 The interplay between hematopoietic cells and bone marrow microenvironment organized by mesenchymal stem cells is important for the maintenance of hematopoiesis. With respect to B cell lymphopoiesis, several constituents of bone marrow microenvironment specific for B cells (“B cell niche”) have been identified, including CXCL12/stromal cell-derived factor-1 (SDF-1)-abundant reticular cells as cellular factors, and CXCL12/SDF-1, interleukin (IL)-7, stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (Flt3-L), and nuclear factor kappa-B ligand (RANKL), as essential humoral factors. However, the precise mechanism through which mesenchymal stem cells in the bone marrow microenvironment support B cell lymphopoiesis, especially the role of transcription factors, remains unknown. We show that the mesenchymal stem cells lacking a transcription factor, CCAAT enhancer binding protein (C/EBP) b, are functionally abnormal, which contribute to the impairment of B cell lymphopoiesis in C/EBPb knockout mice. In C/EBPb knockout mice, the number of B cells, in particular, B220+CD43+ precursor B cells, was significantly decreased in bone marrow compared with that in wild-type littermates (Figure 1A and 1B). As shown in Fig. 1A, the percentage of total B220+ B cells was decreased at 19.1 ± 7.1% in the bone marrow of C/EBPb knockout mice (KO, n = 13) compared to wild-type mice (WT, n = 14, 26.5 ± 7.3%: *P<0.05). The percentage of B220+CD43+ precursor B cells was also decreased at 5.2 ± 1.5% in the bone marrow of C/EBPb knockout mice (KO, n = 13) compared to wild-type mice (WT, n = 14, 7.4 ± 1.6%: **P<0.01). Intriguingly, in vivo bone marrow transplantation experiments demonstrated that the bone marrow cells derived from C/EBPb knockout mice were engrafted in lethally-irradiated (10 Gy) wild-type mice with equivalently B cell recovery compared to the bone marrow cells from normal wild-type mice. Conversely, when normal wild-type c-kit+ Sca-1+ lineages− hematopoietic stem cells (KSL cells) were co-cultured with C/EBPb deficient mesenchymal stem cells in vitro (KO), they showed impaired B cell differentiation compared to the co-culture with normal wild-type mesenchymal stem cells (WT, Figure 1C). Mechanistically, the CXCL12/SDF-1 production by C/EBPb deficient mesenchymal stem cells was reduced compared with that by wild-type mesenchymal stem cells (KO, n = 5, 4.47 ± 1.16 ng/mL; WT, n = 5, 9.90 ± 1.93 ng/mL; **P < 0.01). These results suggest a possibility that abnormal C/EBPƒÀ deficient mesenchymal stem cells in bone marrow microenvironment contribute to impaired B cell lymphopoiesis in C/EBPb knockout mice. We further found that C/EBPb deficient mesenchymal stem cells displayed several functional abnormalities. First, calcium accumulation was significantly reduced in 4 week osteogenesis-inducing cultures of C/EBPb-deficient mesenchymal stem cells compared to cultures of wild-type mesenchymal stem cells. This occurred along with the down-regulated expression of the principal osteogenic master molecule runt-related transcription factor 2 (Runx2). Second, lipid deposition was significantly reduced in 1 week adipogenesis-inducing cultures of C/EBPb-deficient mesenchymal stem cells. The expression of adipogenic markers, including peroxisome proliferator-activated receptor b (PPARb) was significantly reduced in adipogenic cultures of C/EBPb-deficient mesenchymal stem cells compared with cultures of wild-type mesenchymal stem cells Finally, the number of colony-forming unit fibroblast (CFU-F) was higher in the bone marrow of C/EBPb knockout mice than in that of wild-type mice. Collectively, C/EBPb-deficient mesenchymal stem cells have aberrant multi-differentiation capability and increased proliferation activity compared with wild-type mesenchymal stem cells, further supporting that C/EBPb-deficient mesenchymal stem cells were functionally abnormal. Altogether, this work demonstrates that impaired B cell lymphopoiesis in C/EBPb knockout mice is attributed to abnormal mesenchymal stem cells in bone marrow microenvironment, at least in a steady-state, an effect that is due in part to the impaired CXCL12/SDF-1 production. Disclosures: No relevant conflicts of interest to declare.

UV-reflectivity of parafocal eyespot elements on butterfly wings in normal and abnormal specimens

An unusual specimen of Aglais urticae, lacking characteristic UV-reflecting parafocal eyespot elements along the margins of both fore and hind wings, is compared with normal, wild-type specimens. Wing scales, responsible for generating structural coloration, aremissing in the abnormal individual and have been replaced with a type that is typical of pigment-based colours. Other modifications seen in the abnormal specimen include firstly, a distal expansion of a uniformly brown region, that otherwise occupies a proximal position on the hind wings of the wild type, and secondly, the lack of a characteristic orange cross-vein band that runs proximal to the parafocal eyespot elements on the hindwing. The differences in coloration between abnormal and wild type are seen as evidence of a proximal-distal developmental axis (originally proposed by Nijhout 1991) and support a view recently aired by Beldade and Brakefield (2003). It is now clear that studies on butterfly eyespot development must consider not only pigmentcontaining scales, but also the structurallymodified scales responsible for physical colours, i.e. UV reflectivity.

Polygalacturonase Levels Influence Electrolyte Efflux in Tomato Fruit

Tomato fruit transformed with the PG-antisense gene have been shown to exhibit persistent structural competence and extended shelf-life compared with normal, PG-containing lines. In this study, PG-antisense and nontransformed, wild-type fruit were examined for electrolyte efflux trends during ripening and following extended storage at the full-ripe stage. Pericarp disks from PG-antisense fruit showed minimal differences in net electrolyte efflux compared with the normal, wild-type fruit at the mature-green through ripe stages of development. Following extended storage (14 days) of ripe fruit, or in response to storage at chilling (1°C) temperatures, significantly higher (25%–33%) values for proportional electrolyte efflux were observed for wild-type fruit. Additionally, ripe wild-type fruit following extended storage or in response to chilling injury exhibited increased (15%–20%) total soluble electrolytes, particularly in tissues subjected to freeze-thaw versus thermal-disruption. Although PG-antisense fruit do exhibit increases in net electrolyte efflux during ripening, the enhanced efflux and electrolyte generation from wild-type ripe fruit during extended storage was due, in part, to the release of polyelectrolytes originating from pectin hydrolysis. These data may explain the divergence in postharvest performance and structural integrity of PG-antisense and normal, wild-type fruit during post-ripe storage and also suggest that modification of the apoplastic environment resulting from developmental increases in electrolyte efflux can enhance the catalytic activity of PG in vivo.

EMG analysis of harmaline-induced tremor in normal and three strains of mutant mice with Purkinje cell degeneration and the role of the inferior olive

1. The effects of intraperitoneal injections of 10 mg/kg harmaline were tested in normal mice and three strains of cerebellar mutant mice with Purkinje cell degeneration. Ten normal (wild-type) mice (+/+), as well as five lurcher (lc/+), six nervous (nr/nr), and eight Purkinje cell degeneration (pcd/pcd) mutants were implanted with chronic electromyogram (EMG) electrodes in the hamstring and quadriceps muscle groups of the right hindlimb. 2. EMGs were recorded in each of the mice during spontaneous activity before and after intraperitoneal injections of 0.3 ml harmaline (10 mg/kg). Spectral analysis was used to quantify the amplitude and frequency of tremor found in the EMGs after harmaline administration. Normal mice responded to harmaline with strong, continuous 11- to 14-Hz tremor. Mutants from the pcd/pcd strain also reacted with continuous tremor, but of lower amplitude and frequency. In contrast, nr/nr mutants exhibited intermittent paroxysmal tremor lasting for only a few seconds, and lc/+ mutants showed no evidence of tremor whatsoever. 3. In order to detect covert tremor that was possibly not revealed by focal intramuscular EMG recordings, several mutant and normal mice were also tested on a suspended platform to which an accelerometer was attached. The results confirmed the findings from EMG recordings. 4. An incidental observation made during the course of this study was that harmaline tremor disappeared from the normal mouse during swimming and reappeared when the animal was withdrawn from the water. 5. Although Purkinje cells appeared to increase both the depth of modulation and the frequency of tremor, the inhibitory action of the cerebellar cortex does not seem to be essential for the generation of tremor. 6. Parasagittal cerebellar sections of the normal, wild-type mice and the three strains of cerebellar mutant mice of various ages were stained with cresyl violet and examined for Purkinje cell degeneration. Purkinje cell degeneration was found to be complete in the pcd/pcd and lc/+ strains. Although an initial examination of parasagittal sections of the nr/nr strain failed to find any surviving Purkinje cells, further examination of sections cut in the coronal plane revealed small clusters of Purkinje cells in the vermal area of the posterior lobe. 7. The retrograde transport of wheat-germ-agglutinin-conjugated horseradish peroxidase (WGA-HRP) pressure-injected into the cerebellar cortex was used to study the olivocerebellar projections in the wild-type mice and the three strains of cerebellar mutant mice.(ABSTRACT TRUNCATED AT 400 WORDS)

NEUROSPORA TREHALASE AND ITS STRUCTURAL GENE

ABSTRACT We have isolated Neurospora trehalaseless mutants and mapped the trehalase structural gene to linkage group I. The structural gene mutations not only affect thermostability and other characteristics of the enzyme but also affect the production of an inhibitor of the wild-type trehalase. The inhibitor appears to be the mutant trehalase. We suggest that the mutant subunits act as inhibitors by entering into the multimeric forms of the enzyme and altering the ability of the normal wild-type subunits to catalyze the cleavage of trehalose.—Wild type trehalase has been purified to near homogeneity, and its characteristics have been studied. It was purified as a tetramer, with each subunit having a molecular weight of 88,000.—We have studied the regulation of trehalase and found the production of trehalase to be glucose repressible. Cells begin to produce trehalase 60 min after being transferred to glucose-free medium.

MUTATIONS LEADING TO EXPRESSION OF THE CRYPTIC HMR a LOCUS IN THE YEAST SACCHAROMYCES CEREVISIAE

ABSTRACT Mutations leading to expression of the silent HMR a information in Saccharomyces cerevisiae result in sporulation proficiency in mat a 1/MATα diploids. An example of such a mutation is sir5-2, a recessive mutation in the gene SIR5. As expected, haploids carrying the sir5-2 mutation are nonmaters due to the simultaneous expression of HMR a and HMLα, resulting in the nonmating phenotype of an a/α diploid. However, sir5-2/sir5-2 mat a 1/MATα diploids mate as α yet are capable of sporulation. The sir5-2 mutation is unlinked to sir1-1, yet the two mutations do not complement each other: mat a 1/MATα sir5-2/SIR5 SIR1/sir1-1 diploids are capable of sporulation. In this case, recessive mutations in two unlinked genes form a mutant phenotype, in spite of the presence of the normal wild-type alleles.—The PAS1-1 mutation, Provider of a Sporulation function, is a dominant mutation tightly linked to HMR a. PAS1-1 does not affect the mating ability of a strain, yet it allows diploids lacking a functional MAT a locus to sporulate. It is proposed that PAS1-1 leads to partial expression of the otherwise cryptic a1 information at HMR a.