Glutamine synthetase V. Dependence of its sulfhydryl requirement on organic ligands and metal ions

A partially purified preparation of glutamine synthetase (-glutamate: ammonia ligase (ADP), EC 6.3.1.2) has been obtained from rat liver. The enzyme preparation showed no activity in the absence of Mg2+; Mn2+ could not replace Mg2+. Mn2+ exhibited a slight activation of the enzyme at low Mg2+ concentrations, but a pronounced inhibition at high Mg2+ concentrations. The inhibition by a number of metal ions including Fe2+, Fe3+, Co2+, Cu+, Zn2+, Cd2+, and Hg2+ depended on the concentration of a thiol. The enzyme showed a partial requirement for a thiol, which could be abolished by certain organic ligands. Among them the most effective were EDTA, hydroxyethyl-ethylenediaminetriacetate, nitrilotriacetate, and 1,10-phenanthroline.Sodium arsenite inhibited the synthetase only in the presence of an active dithiol such as 2,3-dimercaptopropanol, but the inhibition could be reversed by an excess of the dithiol. p-Chloromercuribenzoate, iodoacetate, o-iodosbenzoate and N-ethylmeleimide exerted different degrees of inhibition on the enzyme. The synthetase was alsi sensitive to formamidine disulfide. Antecedent incubation of the enzyme with the inhibitor greatly enhanced the potency of the inhibitor and consequently required a large excess of 2,3-dimercaptopropanol to restore the enzyme activity.These results have been interpreted to mean that glutamine synthetase may contain a metal ion and a dithiol component.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32113/1/0000163.pd

Transformation and tumorigenicity testing of simian cell lines and evaluation of poliovirus replication

The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals. In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated. Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field

To whom does the law speak? Canvassing a neglected picture of law’s interpretive field

Among the most common strategies underlying the so-called indeterminacy thesis is the following two-step argument: (1) that law is an interpretive practice, and that evidently legal actors more generally hold different (and competing) theories of meaning, which lead to disagreements as to what the law says (that is, as to what the law is); (2) and that, as there is no way to establish the prevalence of one particular theory of meaning over the other, indeterminacy is pervasive in law. In this paper I offer some reflections to resist this trend. In particular I claim that a proper understanding of law as an authoritative communicative enterprise sheds new light on the relation between the functioning of the law and our theories of interpretation, leading to what can be considered a neglected conclusion: the centrality of the linguistic criterion of meaning in our juridical interpretive practices. In the first part of the chapter I discuss speech-act theory in the study of law, assessing its relevance between alternative options. Then I tackle the ‘to whom does the law speak?’ question, highlighting the centrality of lay-people for our juridical practices. Lastly, I examine the consequences of this neglected fact for our interpretive theories

Highly Sensitive In Vitro Methods for Detection of Residual Undifferentiated Cells in Retinal Pigment Epithelial Cells Derived from Human iPS Cells

Human induced pluripotent stem cells (hiPSCs) possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs). These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay): soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR). Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×104 RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research

A Honey Bee Hexamerin, HEX 70a, Is Likely to Play an Intranuclear Role in Developing and Mature Ovarioles and Testioles

Insect hexamerins have long been known as storage proteins that are massively synthesized by the larval fat body and secreted into hemolymph. Following the larval-to-pupal molt, hexamerins are sequestered by the fat body via receptor-mediated endocytosis, broken up, and used as amino acid resources for metamorphosis. In the honey bee, the transcript and protein subunit of a hexamerin, HEX 70a, were also detected in ovaries and testes. Aiming to identify the subcellular localization of HEX 70a in the female and male gonads, we used a specific antibody in whole mount preparations of ovaries and testes for analysis by confocal laser-scanning microscopy. Intranuclear HEX 70a foci were evidenced in germ and somatic cells of ovarioles and testioles of pharate-adult workers and drones, suggesting a regulatory or structural role. Following injection of the thymidine analog EdU we observed co-labeling with HEX 70a in ovariole cell nuclei, inferring possible HEX 70a involvement in cell proliferation. Further support to this hypothesis came from an injection of anti-HEX 70a into newly ecdysed queen pupae where it had a negative effect on ovariole thickening. HEX 70a foci were also detected in ovarioles of egg laying queens, particularly in the nuclei of the highly polyploid nurse cells and in proliferating follicle cells. Additional roles for this storage protein are indicated by the detection of nuclear HEX 70a foci in post-meiotic spermatids and spermatozoa. Taken together, these results imply undescribed roles for HEX 70a in the developing gonads of the honey bee and raise the possibility that other hexamerins may also have tissue specific functions

Protein Crosslinking by Transglutaminase Controls Cuticle Morphogenesis in Drosophila

Transglutaminase (TG) plays important and diverse roles in mammals, such as blood coagulation and formation of the skin barrier, by catalyzing protein crosslinking. In invertebrates, TG is known to be involved in immobilization of invading pathogens at sites of injury. Here we demonstrate that Drosophila TG is an important enzyme for cuticle morphogenesis. Although TG activity was undetectable before the second instar larval stage, it dramatically increased in the third instar larval stage. RNA interference (RNAi) of the TG gene caused a pupal semi-lethal phenotype and abnormal morphology. Furthermore, TG-RNAi flies showed a significantly shorter life span than their counterparts, and approximately 90% of flies died within 30 days after eclosion. Stage-specific TG-RNAi before the third instar larval stage resulted in cuticle abnormality, but the TG-RNAi after the late pupal stage did not, indicating that TG plays a key role at or before the early pupal stage. Immediately following eclosion, acid-extractable protein from wild-type wings was nearly all converted to non-extractable protein due to wing maturation, whereas several proteins remained acid-extractable in the mature wings of TG-RNAi flies. We identified four proteins—two cuticular chitin-binding proteins, larval serum protein 2, and a putative C-type lectin—as TG substrates. RNAi of their corresponding genes caused a lethal phenotype or cuticle abnormality. Our results indicate that TG-dependent protein crosslinking in Drosophila plays a key role in cuticle morphogenesis and sclerotization

Recombinant Vesicular Stomatitis Virus Vaccine Vectors Expressing Filovirus Glycoproteins Lack Neurovirulence in Nonhuman Primates

The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses an individual filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV) GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV) GP; three animals received rVSV-wild type (wt) vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use