GPAT2, a mitochondrial outer membrane protein,in piRNA biogenesis in germline stem cells

piRNA (PIWI-interacting RNA) is a germ cell–specific small RNA in which biogenesis PIWI (P-element wimpy testis) family proteins play crucial roles. MILI (mouse Piwi-like), one of the three mouse PIWI family members, is indispensable for piRNA production, DNA methylation of retrotransposons presumably through the piRNA, and spermatogenesis. The biogenesis of piRNA has been divided into primary and secondary processing pathways; in both of these MILI is involved in mice. To analyze the molecular function of MILI in piRNA biogenesis, we utilized germline stem (GS) cells, which are derived from testicular stem cells and possess a spermatogonial phenotype. We established MILI-null GS cell lines and their revertant, MILI-rescued GS cells, by introducing the Mili gene with Sendai virus vector. Comparison of wild-type, MILI-null, and MILI-rescued GS cells revealed that GS cells were quite useful for analyzing the molecular mechanisms of piRNA production, especially the primary processing pathway. We found that glycerol-3-phosphate acyltransferase 2 (GPAT2), a mitochondrial outer membrane protein for lysophosphatidic acid, bound to MILI using the cells and that gene knockdown of GPAT2 brought about impaired piRNA production in GS cells. GPAT2 is not only one of the MILI bound proteins but also a protein essential for primary piRNA biogenesis.     

Heparin-functionalized thermoresponsive surface

Temperature-dependent regulation of affinity binding between bioactive ligands and their cell membrane receptors is an attractive approach for the dynamic control of cellular adhesion, proliferation, migration, differentiation, and signal transduction. Covalent conjugation of bioactive ligands onto thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-grafted surfaces facilitates the modulation of one-on-one affinity binding between bioactive ligands and cellular receptors by changing temperature. For the dynamic control of the multivalent affinity binding between heparin and heparin-binding proteins, thermoresponsive cell culture surface modified with heparin, which interacts with heparin-binding proteins such as basic fibroblast growth factor (bFGF), has been proposed. Heparin-functionalized thermoresponsive cell culture surface induces (1) the multivalent affinity binding of bFGF in active form and (2) accelerating cell sheet formation in the state of shrunken PIPAAm chains at 37°C. By lowering temperature to 20°C, the affinity binding between bFGF and immobilized heparin is reduced with increasing the mobility of heparin and the swollen PIPAAm chains, leading to the detachment of cultured cells. Therefore, heparin-functionalized thermoresponsive cell culture surface was able to enhance cell proliferation and detach confluent cells as a contiguous cell sheet by changing temperature. A cell cultivation system using heparin-functionalized thermoresponsive cell culture surface is versatile for immobilizing other heparin-binding proteins such as vascular endothelial growth factor, fibronectin, antithrombin III, and hepatocyte growth factor, etc. for tuning the adhesion, growth, and differentiation of various cell species.     

A Novel Cell-Sheet Technology That Achieves Durable Factor VIII Delivery in a Mouse Model of Hemophilia A

Gene- or cell-based therapies aimed at creating delivery systems for coagulation factor VIII (FVIII) protein have emerged as promising options for hemophilia A treatment. However, several issues remain to be addressed regarding the efficacies and adverse events of these new classes of therapies. To improve an existing cell-based therapy involving the subcutaneous transplantation of FVIII-transduced blood outgrowth endothelial cells (BOECs), we employed a novel cell-sheet technology that allows individual dispersed cells to form a thin and contiguous monolayer without traditional bioabsorbable scaffold matrices. Compared to the traditional methodology, our cell-sheet approach resulted in longer-term and 3–5-fold higher expression of FVIII (up to 11% of normal) in recipient hemophilia A mice that lacked a FVIII humoral immune response due to transient immunosuppression with cyclophosphamide. Histological studies revealed that the transplanted BOEC sheets were structured as flat clusters, supporting the long-term expression of therapeutic FVIII in plasma from an ectopic subcutaneous space. Our novel tissue-engineering approach using genetically modified BOEC sheets could aid in development of cell-based therapy that will allow safe and effective in vivo delivery of functional FVIII protein in patients with hemophilia A.     

Functionally Cloned pdrM from Streptococcus pneumoniae Encodes a Na+ Coupled Multidrug Efflux Pump

Multidrug efflux pumps play an important role as a self-defense system in bacteria. Bacterial multidrug efflux pumps are classified into five families based on structure and coupling energy: resistance−nodulation−cell division (RND), small multidrug resistance (SMR), major facilitator (MF), ATP binding cassette (ABC), and multidrug and toxic compounds extrusion (MATE). We cloned a gene encoding a MATE-type multidrug efflux pump from Streptococcus pneumoniae R6, and designated it pdrM. PdrM showed sequence similarity with NorM from Vibrio parahaemolyticus, YdhE from Escherichia coli, and other bacterial MATE-type multidrug efflux pumps. Heterologous expression of PdrM let to elevated resistance to several antibacterial agents, norfloxacin, acriflavine, and 4′,6-diamidino-2-phenylindole (DAPI) in E. coli KAM32 cells. PdrM effluxes acriflavine and DAPI in a Na⁺- or Li⁺-dependent manner. Moreover, Na⁺ efflux via PdrM was observed when acriflavine was added to Na⁺-loaded cells expressing pdrM. Therefore, we conclude that PdrM is a Na⁺/drug antiporter in S. pneumoniae. In addition to pdrM, we found another two genes, spr1756 and spr1877,that met the criteria of MATE-type by searching the S. pneumoniae genome database. However, cloned spr1756 and spr1877 did not elevate the MIC of any of the investigated drugs. mRNA expression of spr1756, spr1877, and pdrM was detected in S. pneumoniae R6 under laboratory growth conditions. Therefore, spr1756 and spr1877 are supposed to play physiological roles in this growth condition, but they may be unrelated to drug resistance.     

Cascade screening and genetic diagnosis of familial hypercholesterolemia in clusters of the Southeastern region from Brazil

Familial hypercholesterolemia (FH) is an autosomal dominant genetic disease characterized by high levels of low-density lipoprotein-cholesterol (LDLc), associated to premature cardiovascular disease. The detection of the variants related to FH is important to improve the early diagnosis in probands / index-cases (ICs) and their relatives. We included ICs with FH and their relatives, living in a small region of Minas Gerais state-Brazil, which were classified according to Dutch Lipid Clinic Network Criteria (DLCNC) and submitted to sequencing of genes related to FH (LDLR, APOB, PCSK9, LDLRAP1, LIPA, STAP1, APOE, ABCG5 e ABCG8). In a total of 143 subjects (32 ICs and 111 relatives), eight variants were identified in 91 individuals. From these variants, five were in LDLR [p.(Asp224Asn), p.(Ser854Gly), p.(Cys34Arg), p.(Asp601His), deletion of exon15 in LDLR)], one in APOB [p.(Met499Val)], one in PCSK9 [p.(Arg237Trp)] and one in APOE [p.(Pro28Leu)] genes. The variants were detected in 100% of those subjects classified as definitive, 87% as probable and 69% as possible FH cases based on DLCNC. The LDLc level was higher in individuals with corneal arch and xanthomas or xanthelasmas, as well as in pathogenic or probably pathogenic variants carriers. This study showed higher frequency of LDLR gene variants compared to other genes related to LDL metabolism in individuals with FH in Minas Gerais – Brazil and the presence of FH in relatives without previous diagnosis. Our data reinforce the importance of molecular and clinical evaluation of FH relatives in order to early diagnosis the FH, as well as cardiovascular diseases prevention.

     

In vitro regulatory models for systems biology

The reductionist approach has revolutionized biology in the past 50 years. Yet its limits are being felt as the complexity of cellular interactions is gradually revealed by high-throughput technology. In order to make sense of the deluge of “omic data”, a hypothesis-driven view is needed to understand how biomolecular interactions shape cellular networks. We review recent efforts aimed at building in vitro biochemical networks that reproduce the flow of genetic regulation. We highlight how those efforts have culminated in the rational construction of biochemical oscillators and bistable memories in test tubes. We also recapitulate the lessons learned about in vivo biochemical circuits such as the importance of delays and competition, the links between topology and kinetics, as well as the intriguing resemblance between cellular reaction networks and ecosystems.     

Identification of a cyanobacterial CRR6 protein,Slr1097, required for efficient assembly of NDH‐1 complexes in Synechocystis sp. PCC 6803

Despite significant progress in clarifying the subunit compositions and functions of the multiple NADPH dehydrogenase (NDH‐1) complexes in cyanobacteria, the subunit maturation and assembly of their NDH‐1 complexes are poorly understood. By transformation of wild‐type cells with a transposon‐tagged library, we isolated three mutants of Synechocystis sp. PCC 6803 defective in NDH‐1‐mediated cyclic electron transfer and unable to grow under high light conditions. All the mutants were tagged in the same slr1097 gene, encoding an unknown protein that shares significant homology with the Arabidopsis protein chlororespiratory reduction 6 (CRR6). The slr1097 product was localized in the cytoplasm and was required for efficient assembly of NDH‐1 complexes. Analysis of the interaction of Slr1097 with 18 subunits of NDH‐1 complexes using a yeast two‐hybrid system indicated a strong interaction with NdhI but not with other Ndh subunits. Absence of Slr1097 resulted in a significant decrease of NdhI in the cytoplasm, but not of other Ndh subunits including NdhH, NdhK and NdhM; the decrease was more evident in the cytoplasm than in the thylakoid membranes. In the ?slr1097 mutant, NdhH, NdhI, NdhK and NdhM were hardly detectable in the NDH‐1M complex, whereas almost half the wild‐type levels of these subunits were present in NDH‐1L complex; similar results were observed in the NdhI‐less mutant. These results suggest that Slr1097 is involved in the maturation of NdhI, and that assembly of the NDH‐1M complex is strongly dependent on this factor. Maturation of NdhI appears not to be crucial to assembly of the NDH‐1L complex.     

Modification of Proteinase Inhibitor II in Adzuki Beans during Germination

We investigated the effects of compounds isolated from a methanolic extract of rose hips on melanin biosynthesis in B16 mouse melanoma cells and the possible mechanisms responsible for the inhibition of melanin biosynthesis. We found that, among the isolated compounds, quercetin was a particularly potent melanogenesis inhibitor. To reveal the mechanism for this inhibition, the effects on tyrosinase of B16 mouse melanoma were measured. Quercetin decreased the intracellular tyrosinase activity as well as the tyrosinase activity in a cell culture-free system. We also examined the cellular level of tyrosinase protein and found that quercetin dose-dependently inhibited tyrosinase protein expression. We consider from these results that the inhibition of melanogenesis by quercetin was due to the inhibition of both tyrosinase activity and of the protein expression.     

Estimation of Aeration and Agitation Conditions in Respect to Oxygen Supply and Ventilation

In inosine fermentation, the yield of the product was closely related to the partial pressure of carbon dioxide in the culture system rather than the bicarbonate ion concentration in the culture liquid. The inhibitory effect of carbon dioxide was restored by the methods of reducing the partial pressure of carbon dioxide lower than a certain level. Both ventilation and chemical absorption were applicable for the restoration of the carbon dioxide inhibition, but ventilation had great advantages over the other method from an industrial view-point. In the estimation of aeration-agitation conditions for the scale-up of submerged fermentation in which carbon dioxide was inhibitory, the rate of aeration was to be established to make sufficient ventilation and overcome this inhibition. A scheme for the procedure for the estimation of aeration-agitation conditions under the consideration of influences of carbon dioxide on submerged fermentation was proposed in this paper.     

Studies on the Bacteria Isolated from Wine

The growth factor for malo-lactic fermentation bacteria was partially purified from tomato juice, and some properties of the factor were examined. The factor was comparatively a low molecule, thermo-stable and insoluble in nonpolar organic solvents. The purified factor fraction showed positive reaction with Molisch-, tetrazolium- and Nessler-reagents, suggesting the existence of sugar and nitrogen as the chemical components. The treatment with cellobiase or cellulase-II resulted in the inactivation of the factor and simultaneously released the sugar constituent composed of glucose from the factor. These results indicated that the glucose residue might exist as the β-1,4-glucosidic linkage in the growth factor.