The interplay between SUCLA2, SUCLG2, and mitochondrial DNA depletion

SUCLA2-related mitochondrial DNA (mtDNA) depletion syndrome is a result of mutations in the β subunit of the ADP-dependent isoform of the Krebs cycle succinyl-CoA synthase (SCS). The mechanism of tissue specificity and mtDNA depletion is elusive but complementation by the GDP-dependent isoform encoded by SUCLG2, and the association with mitochondrial nucleoside diphosphate kinase (NDPK), is a plausible link. We have investigated this relationship by studying SUCLA2 deficient fibroblasts derived from patients and detected normal mtDNA content and normal NDPK activity. However, knockdown of SUCLG2 by shRNA in both patient and control fibroblasts resulted in a significant decrease in mtDNA amount, decreased NDPK and cytochrome c oxidase activities, and a marked growth impairment. This suggests that, SUCLG2, to a higher degree than SUCLA2, is crucial for mtDNA maintenance and that mitochondrial NDPK is involved. Although results pertain to a cell culture system, the findings might explain the pathomechanism and tissue specificity in mtDNA depletion caused by defective SUCLA2.     

Identification of a negative regulatory region for the exchange activity and characterization of T332I mutant of Rho guanine nucleotide exchange factor 10 (ARHGEF10)

The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1-332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant.     

Identification of G Protein-Coupled Receptors (GPCRs) in Primary Cilia and Their Possible Involvement in Body Weight Control

Primary cilia are sensory organelles that harbor various receptors such as G protein-coupled receptors (GPCRs). We analyzed subcellular localization of 138 non-odorant GPCRs. We transfected GPCR expression vectors into NIH3T3 cells, induced ciliogenesis by serum starvation, and observed subcellular localization of GPCRs by immunofluorescent staining. We found that several GPCRs whose ligands are involved in feeding behavior, including prolactin-releasing hormone receptor (PRLHR), neuropeptide FF receptor 1 (NPFFR1), and neuromedin U receptor 1 (NMUR1), localized to the primary cilia. In addition, we found that a short form of dopamine receptor D2 (DRD2S) is efficiently transported to the primary cilia, while a long form of dopamine receptor D2 (DRD2L) is rarely transported to the primary cilia. Using an anti-Prlhr antibody, we found that Prlhr localized to the cilia on the surface of the third ventricle in the vicinity of the hypothalamic periventricular nucleus. We generated the Npy2r-Cre transgenic mouse line in which Cre-recombinase is expressed under the control of the promoter of Npy2r encoding a ciliary GPCR. By mating Npy2r-Cre mice with Ift80 flox mice, we generated Ift80 conditional knockout (CKO) mice in which Npy2r-positive cilia were diminished in number. We found that Ift80 CKO mice exhibited a body weight increase. Our results suggest that Npy2r-positive cilia are important for body weight control.     

A study of accessible motifs and RNA folding complexity.

mRNA molecules are folded in the cells and therefore many of their substrings may actually be inaccessible to protein and microRNA binding. The need to apply an accessibility criterion to the task of genome-wide mRNA motif discovery raises the challenge of overcoming the core O(n(3)) factor imposed by the time complexity of the currently best known algorithms for RNA secondary structure prediction. We speed up the dynamic programming algorithms that are standard for RNA folding prediction. Our new approach significantly reduces the computations without sacrificing the optimality of the results, yielding an expected time complexity of O(n(2) psi(n)), where psi(n) is shown to be constant on average under standard polymer folding models. A benchmark analysis confirms that in practice the runtime ratio between the previous approach and the new algorithm indeed grows linearly with increasing sequence size. The fast new RNA folding algorithm is utilized for genome-wide discovery of accessible cis-regulatory motifs in data sets of ribosomal densities and decay rates of S. cerevisiae genes and to the mining of exposed binding sites of tissue-specific microRNAs in A. thaliana.     

Atherogenic low density lipoprotein phenotype in long-term survivors of childhood acute lymphoblastic leukemia

Survivors of childhood acute lymphoblastic leukemia (ALL) have an increased risk of cardiovascular disease. Small density lipoproteins are atherogenic but have not been studied in this population. We conducted a cross-sectional analysis of 110 ALL survivors (mean age, 24.3 years) to determine prevalence of small dense LDL (pattern B) phenotype in ALL survivors and identify associated factors. Lipid subfractions were measured using Vertical Auto Profile-II. Participants with greater than 50% of LDL-cholesterol (LDL-c) in small dense LDL fractions (LDL₃₊₄) were classified as LDL pattern B. Visceral and subcutaneous adipose tissue (VAT, SAT) volumes were also measured by computed tomography. While the mean LDL-c level of ALL survivors was 108.7 ± 26.8 mg/dl, 36% (40/110) of survivors had atherogenic LDL pattern B. This pattern was more common in males (26/47; 55%) than in females (14/63; 22%, P = 0.001) and more common in survivors treated with cranial radiotherapy (15/33; 45%) than in those who were treated with chemotherapy alone (25/77; 33%; P = 0.04, adjusted for age, gender, history of hypertension, and smoking history). VAT was associated with atherogenic lipids: LDL pattern B and LDL₃₊₄ levels. This association was independent of other measures of body fat. We conclude that a substantial proportion of ALL survivors had an atherogenic LDL phenotype despite normal mean LDL-c levels. An atherogenic LDL phenotype may contribute to the increase in cardiovascular mortality and morbidity in this population.     

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.Download video file.^{(53M, mov)}     

Assessment of cell engineering strategies for improved therapeutic protein production in CHO cells

Recombinant glycoprotein therapeutics have proven to be invaluable pharmaceuticals for the treatment of various diseases. Chinese hamster ovary (CHO) cells are widely used in industry for the production of these proteins. Several strategies for engineering CHO cells for improved protein production have been tried with considerable results. The focus has mainly been to increase the specific productivity and to extend the culture longevity by preventing programmed cell death. These CHO cell engineering strategies, particularly those developed in Korea, are reviewed here.     

Effect of Bcl‐xL overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient‐deprived condition

Upon nutrient deprivation during culture, recombinant Chinese hamster ovary (rCHO) cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. To investigate the effect of Bcl‐x_L overexpression on apoptosis and autophagy in rCHO cells, an erythropoietin (EPO)‐producing rCHO cell line with regulated Bcl‐x_L overexpression (EPO‐off‐Bcl‐x_L) was established using the Tet‐off system. The expression level of Bcl‐x_L in EPO‐off‐Bcl‐x_L cells was tightly regulated by doxycycline in a dose‐dependent manner. Bcl‐x_L overexpression enhanced cell viability and extended culture longevity in batch culture. Upon nutrient depletion in the later stage of batch culture, Bcl‐x_L overexpression suppressed apoptosis by inhibiting the activation of caspase‐3 and ‐7. Simultaneously, Bcl‐x_L overexpression also delayed autophagy, characterized by LC3‐II accumulation. Immunoprecipitation analysis with a Flag‐tagged Bcl‐x_L revealed that Bcl‐x_L interacts with Bax and Bak, essential mediators of caspase‐dependent apoptosis, as well as with Beclin‐1, an essential mediator of autophagy, and may inhibit their pro‐cell death function. Taken together, it was found that Bcl‐x_L overexpression inhibits both apoptosis and autophagy in rCHO cell culture. Biotechnol. Bioeng. 2009;103: 757–766. © 2009 Wiley Periodicals, Inc.