24 research outputs found
Asynchronous nuclear division cycles in multinucleated cells
No full textSynchronous mitosis is common in multinucleated cells. We analyzed a unique asynchronous nuclear division cycle in a multinucleated filamentous fungus, Ashbya gossypii. Nuclear pedigree analysis and observation of GFP-labeled spindle pole bodies demonstrated that neighboring nuclei in A. gossypii cells are in different cell cycle stages despite close physical proximity. Neighboring nuclei did not differ significantly in their patterns of cyclin protein localization such that both G1 and mitotic cyclins were present regardless of cell cycle stage, suggesting that the complete destruction of cyclins is not occurring in this system. Indeed, the expression of mitotic cyclin lacking NH(2)-terminal destruction box sequences did not block cell cycle progression. Cells lacking AgSic1p, a predicted cyclin-dependent kinase (CDK) inhibitor, however, showed aberrant multipolar spindles and fragmented nuclei that are indicative of flawed mitoses. We hypothesize that the continuous cytoplasm in these cells promoted the evolution of a nuclear division cycle in which CDK inhibitors primarily control CDK activity rather than oscillating mitotic cyclin proteins
Limited functional redundancy and oscillation of cyclins in multinucleated Ashbya gossypii fungal cells
No full textCyclin protein behavior has not been systematically investigated in multinucleated cells with asynchronous mitoses. Cyclins are canonical oscillating cell cycle proteins, but it is unclear how fluctuating protein gradients can be established in multinucleated cells where nuclei in different stages of the division cycle share the cytoplasm. Previous work in A. gossypii, a filamentous fungus in which nuclei divide asynchronously in a common cytoplasm, demonstrated that one G1 and one B-type cyclin do not fluctuate in abundance across the division cycle. We have undertaken a comprehensive analysis of all G1 and B-type cyclins in A. gossypii to determine whether any of the cyclins show periodic abundance across the cell cycle and to examine whether cyclins exhibit functional redundancy in such a cellular environment. We localized all G1 and B-type cyclins and notably found that only AgClb5/6p varies in subcellular localization during the division cycle. AgClb5/6p is lost from nuclei at the meta-anaphase transition in a D-box-dependent manner. These data demonstrate that efficient nuclear autonomous protein degradation can occur within multinucleated cells residing in a common cytoplasm. We have shown that three of the five cyclins in A. gossypii are essential genes, indicating that there is minimal functional redundancy in this multinucleated system. In addition, we have identified a cyclin, AgClb3/4p, that is essential only for sporulation. We propose that the cohabitation of different cyclins in nuclei has led to enhanced substrate specificity and limited functional redundancy within classes of cyclins in multinucleated cells
Pre-existing antibodies to candidate gene therapy vectors (adeno-associated vector serotypes) in domestic cats
No full textAdeno-associated virus (AAV) vectors represent promising candidates for gene therapy; however, pre-existing neutralizing antibodies (NAb) may reduce AAV vector delivery efficiency. In this study, the presence of AAV NAb was investigated in cats, which serve as a larger and outbred animal model for the prediction of gene therapy outcomes in humans but also in cats.Serum/plasma samples from 230 client-owned Swiss cats and 20 specified pathogen-free cats were investigated for NAb to AAV1, AAV2, AAV5, AAV6, AAV7, AAV8 and AAV9 using in vitro transduction inhibition and a beta-galactosidase assay. NAb to all tested AAV serotypes were found. Of the client-owned cats, 53% had NAb to one or more of the AAV serotypes. NAb (≥1:10) were found at frequencies of 5% (AAV6) to 28% (AAV7). The highest titers were found against AAV7 (≥1:160). The NAb prevalence to AAV2, AAV7 and AAV9 differed geographically. Regarding titers ≥1:10 against single AAV serotypes, age, breed and sex of the cats were not associated with the NAb prevalence. Cats with titers ≥1:20 against AAV2 and titers ≥1:40 against AAV7 were significantly younger than cats with low/no titers, and purebred cats were significantly more likely than non-purebred cats to have NAb to AAV2 (≥1:40). Additionally, regarding NAb to all AAV combined, female cats were significantly more likely than male cats to have NAb titers ≥1:40. Preliminary data using AAV-DJ indicated that less pre-existing NAb to the hybrid AAV-DJ can be expected compared to the wild-type AAV serotypes. AAV NAb will need to be taken into account for future in vivo gene therapy studies in cats.</div
GAPDH pseudogenes and the quantification of feline genomic DNA equivalents
No full textQuantitative real-time PCR (qPCR) is broadly used to detect and quantify nucleic acid targets. In order to determine cell copy number and genome equivalents, a suitable reference gene that is present in a defined number in the genome is needed, preferably as a single copy gene. For most organisms, a variable number of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pseudogenes have been reported. However, it has been suggested that a single-copy of the GAPDH pseudogene is present in the feline genome and that a GAPDH assay can therefore be used to quantify feline genomic DNA (gDNA). The aim of this study was to determine whether one or more GAPDH pseudogenes are present in the feline genome and to provide a suitable alternative qPCR system for the quantification of feline cell copy number and genome equivalents. Bioinformatics and sequencing results revealed that not just one but several closely related GAPDH-like sequences were present in the cat genome. We thus identified, developed, optimized, and validated an alternative reference gene assay using feline albumin (fALB). Our data emphasize the need for an alternative reference gene, apart from the GAPDH pseudogene, for the normalization of gDNA levels. We recommend using the fALB qPCR assay for future studies
Geographic origin of cats with NAb against AAV in Switzerland.
No full textNAb against A) AAV1, B) AAV2, C) AAV5, D) AAV6, E) AAV7, F) AAV8, and G) AAV9 are shown. Dots represent samples with positive NAb against an AAV serotype. Higher color intensity refers to higher positive NAb titers, with 1:160 being the darkest. Black dots represent geographic locations where more than one sample with a positive NAb was found. In this case, all positive samples are clustered around the black dot.</p
Pre-existing antibodies to candidate gene therapy vectors (adeno-associated vector serotypes) in domestic cats
No full textAdeno-associated virus (AAV) vectors represent promising candidates for gene therapy; however, pre-existing neutralizing antibodies (NAb) may reduce AAV vector delivery efficiency. In this study, the presence of AAV NAb was investigated in cats, which serve as a larger and outbred animal model for the prediction of gene therapy outcomes in humans but also in cats.Serum/plasma samples from 230 client-owned Swiss cats and 20 specified pathogen-free cats were investigated for NAb to AAV1, AAV2, AAV5, AAV6, AAV7, AAV8 and AAV9 using in vitro transduction inhibition and a beta-galactosidase assay. NAb to all tested AAV serotypes were found. Of the client-owned cats, 53% had NAb to one or more of the AAV serotypes. NAb (≥1:10) were found at frequencies of 5% (AAV6) to 28% (AAV7). The highest titers were found against AAV7 (≥1:160). The NAb prevalence to AAV2, AAV7 and AAV9 differed geographically. Regarding titers ≥1:10 against single AAV serotypes, age, breed and sex of the cats were not associated with the NAb prevalence. Cats with titers ≥1:20 against AAV2 and titers ≥1:40 against AAV7 were significantly younger than cats with low/no titers, and purebred cats were significantly more likely than non-purebred cats to have NAb to AAV2 (≥1:40). Additionally, regarding NAb to all AAV combined, female cats were significantly more likely than male cats to have NAb titers ≥1:40. Preliminary data using AAV-DJ indicated that less pre-existing NAb to the hybrid AAV-DJ can be expected compared to the wild-type AAV serotypes. AAV NAb will need to be taken into account for future in vivo gene therapy studies in cats
Linkage of seroreactivity (R<sub>AB</sub>) between AAV serotypes in client-owned cats in Switzerland (NAb titer: 1:10).
No full textLinkage of seroreactivity (RAB) between AAV serotypes in client-owned cats in Switzerland (NAb titer: 1:10).</p
Overview of the prevalence (in %) of NAb to AAV in various animal species.
No full textOverview of the prevalence (in %) of NAb to AAV in various animal species.</p
Prevalence of pre-existing NAb against AAV serotypes in client-owned domestic cats from Switzerland depicted according to titers.
No full textThe percentage of NAb against different AAV serotypes is shown for serum dilutions of ≥1:10; ≥1:20, ≥1:40, ≥1:80 and ≥1:160. NAb against AAV7, AAV8 and AAV9 were the most prevalent; NAb against AAV1, AAV5 and AAV6 were the least prevalent.</p
List of AAV vectors, multiplicity of infection (MOI) values and cell lines used for the transduction inhibition assay.
No full textList of AAV vectors, multiplicity of infection (MOI) values and cell lines used for the transduction inhibition assay.</p
