Flow cytometry and factor analysis evaluation of confocal image sequences of morphologic and functional changes occurring at the mitochondrial level during 7-ketocholesterol-induced cell death

OBJECTIVE: To analyze functional and morphologic alterations that occur at the mitochondrial level by flow cytometry and laser scanning confocal microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Under treatment of U937 cells with 7-ketocholesterol, functional alterations that occur at the mitochondrial level (especially loss of transmembrane mitochondrial potential [delta psi m]) were assessed with 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and mitotracker red (CMXRos), whereas morphologic changes were analyzed with nonyl acridine orange (NAO). By flow cytometry, these different dyes were excited at 488 nm, whereas on CLSM, excitation of NAO and CMXRos was performed by lines of an argon laser. By CLSM, spectral sequences were performed to characterize NAO and CMXRos. FAMIS was used to transform the image sequences in factor images. RESULTS: By flow cytometry, rapid loss of delta psi m induced by 7-ketocholesterol was detected with both DiOC6(3) and CMXRos, which gave similar results. Morphologic alterations of mitochondria were revealed with NAO. The factor images obtained from confocal image sequences confirmed these results. CONCLUSION: The simultaneous use of NAO, CMXRos and FAMIS constitutes a new method to detect morphologic and functional alterations occurring at the mitochondrial level during cell death.     

Identification of two novel ACTH-responsive genes encoding manganese-dependent superoxide dismutase (SOD2) and the zinc finger protein TIS11b [tetradecanoyl phorbol acetate (TPA)-inducible sequence 11b

ACTH is the major trophic factor regulating and maintaining adrenocortical function, affecting such diverse processes as steroidogenesis, cell proliferation, cell migration, and cell survival. We used differential display RT-PCR to identify genes that are rapidly induced by ACTH in the bovine adrenal cortex. Of 42 PCR products differentially amplified from primary cultures of bovine adrenocortical cells treated with 10 nM ACTH, six identified mRNAs that were confirmed by Northern blot analysis to be induced by ACTH. Four of these amplicons encoded noninformative repetitive sequences. Of the other two sequenced amplicons, one encoded a partial sequence for mitochondrial manganese-dependent superoxide dismutase (SOD2), an enzyme that is likely to protect adrenocortical cells from the cytotoxic effects of radical oxygen species generated during steroid biosynthesis. The second was identified as TIS11b (phorbol-12-myristate-13-acetate-inducible sequence 11b)/ERF-1/cMG, a member of the CCCH double-zinc finger protein family. SOD2 induction by ACTH was independent of extracellular steroid concentration or oxidative stress. SOD2 and TIS11b mRNA expressions were rapidly induced by ACTH, reaching a maximal level after 8 h and 3 h of treatment, respectively. These ACTH effects were mimicked by forskolin but appeared independent of cortisol secretion. Upon ACTH treatment, induction of TIS11b expression closely followed the previously characterized peak of vascular endothelial growth factor (VEGF) expression. Transfection of a TIS11b expression plasmid into 3T3 fibroblasts induced a decrease in the expression of a reporter gene placed upstream of the VEGF 3'-untranslated region, indicating that TIS11b may be an important regulator of VEGF expression through interaction with its 3'-untranslated region.     

Localization of 113 anchor loci in pigs: improvement of the comparative map for humans, pigs, and goats

In total, 113 genes that have already been located in humans and goats were cytogenetically mapped in pigs. For this purpose, 165 gene-containing bacterial artificial chromosomes (BACs) isolated in goats were used in heterologous fluorescent in situ hybridization on porcine chromosomes. Among them, 113 (or 69%) gave clear and specific signals, and 52 did not work in heterologous conditions. These localizations are a significant contribution to development of the porcine gene map and also to the comparative map for humans and pigs. They allowed us to specify the information obtained by Zoo-FISH while taking the gene order into account; the number of conserved fragments detected for human and pig chromosomes reached 84. The average size of conserved fragments could be estimated at 33 cM. As these genes had already been mapped in goats, the comparison was extended to ruminants. The previous results obtained in this species, suggesting a correlation between human chromosome abnormalities and evolutionary breakpoints, were confirmed in pigs. Received: 22 July 1999 / Accepted: 3 December 1999     

Construction of a horse BAC library and cytogenetical assignment of 20 type I and type II markers

A horse BAC library was constructed with about 40, 000 clones and mean insert size of 110 kb representing a 1.5 genome equivalent coverage and a probability of finding a single sequence of 0.75. It was characterized by PCR screening of about 130 sequences of horse microsatellites and exonic gene sequences retrieved from databases. BACs containing 8 microsatellites and 12 genes were subsequently localized by fluorescent in situ hybridization (FISH) on chromosomes. Two linkage groups were newly assigned to chromosomes: LG2 to ECA3 and LG5 to ECA24, and five linkage groups were also oriented—LG3, LG4, LG5, LG8, and LG12—leaving only three groups unassigned. This work showed how this library makes an integrated map a realistic objective for the near future and how it can make comparative mapping more efficient in a search for candidate genes of interest. Received: 9 January 1998 / Accepted: 13 April 1998     

A novel variant in DMXL2 gene is associated with autosomal dominant non-syndromic hearing impairment (DFNA71) in a Cameroonian family

Approximately half of congenital hearing impairment cases are inherited, with non-syndromic hearing impairment (NSHI) being the most frequent clinical entity of genetic hearing impairment cases. A family from Cameroon with NSHI was investigated by performing exome sequencing using DNA samples obtained from three family members, followed by direct Sanger sequencing in additional family members and controls participants. We identified an autosomal dominantly inherited novel missense variant [NM_001174116.2:c.918G>T; p.(Q306H)] in DMXL2 gene (MIM:612186) that co-segregates with mild to profound non-syndromic sensorineural hearing impairment . The p.(Q306H) variant which substitutes a highly conserved glutamine residue is predicted deleterious by various bioinformatics tools and is absent from several genome databases. This variant was also neither found in 121 apparently healthy controls without a family history of hearing impairment , nor 112 sporadic NSHI cases from Cameroon. There is one previous report of a large Han Chinese NSHI family that segregates a missense variant in DMXL2. The present study provides additional evidence that DMXL2 is involved in hearing impairment etiology, and we suggest DMXL2 should be considered in diagnostic hearing impairment panels.     

Wie sich Oasen‐Bananen gegen ihre Feinde wehren. Uralter Toleranzmechanismus im Oman entdeckt

Until recently little was known about the crop diversity in the Sultanate of Oman, situated at the NE tip of the Arabian Peninsula. Interdisciplinary research in the often millenia‐old oases provide evidence for their role as reservoirs for plant genetic resources of ancient varieties of wheat and banana. Two newly discovered banana clones show a highly efficient biochemical defense mechanisms against some of the most devastating pests and diseases of banana. If these mechanisms can be properly understood and exploited in breeding programs, may have major impact on the commercial production of edible banana.     

Home-range Use by a Large Horde of Wild <Emphasis Type="Italic">Mandrillus sphinx</Emphasis>

The predicted relationship between home-range size and group mass in primates developed by Clutton-Brock and Harvey (1977) has proved extremely robust in describing the use of space by most primate species. However, mandrills (Mandrillus sphinx) are now known to have an extreme group mass in the wild, far larger than that of the species used originally to generate that relationship, and so it was unknown whether this relationship would be robust for this species. We investigated the home-range size and use of a wild horde of ca. 700 mandrills in Lopé National Park, Gabon, using radiotelemetry. The total area the horde used over a 6-yr period [100% minimum convex polygon (MCP)] was 182 km², including 89 km² of suitable forest habitat. Mandrills used gallery forests and isolated forest fragments with high botanical diversity far more intensively that the continuous forest and completely avoided savanna and marsh. Peeled polygons and fixed kernel contours revealed multiple centres of use, with the horde spending more than half its time in <10% of the total documented range, typical of a frugivore using a patchy environment. Home-range size and internal structure varied considerably between years, but total home range fitted the predicted relationship between group mass and home range size, despite being an outlier to the dataset. We discuss the conservation implications of the species’ space requirements, in light of current pressures on land use in their range.     

Highly efficient deletion of FUT8 in CHO cell lines using zinc‐finger nucleases yields cells that produce completely nonfucosylated antibodies

IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily α1,6‐fucosylated, a modification that reduces antibody‐dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a α1,6‐fucosyltransferase. FUT8^?/? CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein‐production cell lines has prevented the widespread adoption of FUT8^?/? cells as hosts for antibody production. We have created zinc‐finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8^?/? CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN‐treated cells can be directly selected to give FUT8^?/? cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN‐derived FUT8^?/? cell lines were as transfectable as wild‐type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild‐type and ZFN‐generated FUT8^?/? cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN‐generated FUT8^?/? cell lines was fourfold higher than in the best‐producing clones of FUT8^?/? cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN‐mediated transfer of the Fut8? phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies. Biotechnol. Bioeng. 2010;106: 774–783. © 2010 Wiley Periodicals, Inc.