Analysis of Neurotrophin/Receptor Interactions with a gD-Flag-Modified Quantitative Kinase Receptor Activation (gD.KIRA) Enzyme-Linked Immunosorbent Assay

A rapid, sensitive, and high-capacity assay has been developed to quantify ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a “kinase receptor activation” or KIRA-ELISA, utilizes two separate microtiter plates, one for cell culture and ligand stimulation, and the other for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of neurotrophin-induced trkA, trkB, or trkC activation. It utilizes a trkA, trkB, or trkC receptor fused with a 26-amino-acid polypeptide flag derived from HSV glycoprotein D (gD.trkA, B, or C, respectively) on the amino-terminus, stably transfected into CHO cells. Stimulated receptors were solubilized with Triton X-100 buffer and then captured in ELISA wells coated with gD-specific mAb. The degree of receptor autophosphorylation was quantified by anti-phosphotyrosine ELISA. Reproducible standard curves were generated with an EC₅₀of approximately 16 ng/ml NGF for gD.trkA KIRA, 11 ng/ml for NT4/5 and 20 ng/ml for BDNF in gD.trkB KIRA, and 9.4 ng/ml for NT3 in gD.trkC KIRA. When the gD.trkA KIRA assay was used to quantify serum NGF or NT3 following administration to rats, the assay agreed well with currently existing ELISA assays. When the gD.trkA KIRA assay was used to test several NGF variants, as well as NGF stability samples, the capacity of the assay to quantify ligand bioactivity compared well with the more widely used radioreceptor binding and PC 12 cell survival assays. The gD.trk KIRA assays show great potential as rapid bioassays, capable of quantitative, consistent, and stability indicating analyses.     

Phosphate incorporation into "nuclear" residual proteins of goose erythrocytes

Minor fractions of “residual proteins” from erythroblast-enriched avian erythroid cells were found to incorporate significantly larger amounts of radiophosphate than similar fractions from mature erythrocytes. This higher level of incorporation could not be detected in the bulk, unfractionated residual proteins from cell populations, respectively, enriched in erythroblasts, reticulocytes, or mature erythrocytes, probably because of variable amounts of phosphate-free protein. It was confirmed that when avian erythroid cells incorporated radiophosphate, specific activities of chromosomal “residual proteins” were orders of magnitude greater than those of histones, although levels of alkali-labile phosphate were only a fewfold higher.     

Mycoplasma infection and hypoxia initiate succinate accumulation and release in the VM-M3 cancer cells

Succinate is known to act as an inflammatory signal in classically activated macrophages through stabilization of HIF-1α leading to IL-1β production. Relevant to this, hypoxia is known to drive succinate accumulation and release into the extracellular milieu. The metabolic alterations associated with succinate release during inflammation and under hypoxia are poorly understood. Data are presented showing that Mycoplasma arginini infection of VM-M3 cancer cells enhances the Warburg effect associated with succinate production in mitochondria and eventual release into the extracellular milieu. We investigated how succinate production and release was related to the changes of other soluble metabolites, including itaconate and 2-HG. Furthermore, we found that hypoxia alone could induce succinate release from the VM-M3 cells and that this could occur in the absence of glucose-driven lactate production. Our results elucidate metabolic pathways responsible for succinate accumulation and release in cancer cells, thus identifying potential targets involved in both inflammation and hypoxia. This article is part of a Special Issue entitled 20th European Bioenergetics Conference, edited by László Zimányi and László Tretter.     

Exome sequencing reveals a <Emphasis Type="Italic">nebulin</Emphasis> nonsense mutation in a dog model of nemaline myopathy

Nemaline myopathy (NM) is a congenital muscle disorder associated with muscle weakness, hypotonia, and rod bodies in the skeletal muscle fibers. Mutations in 10 genes have been implicated in human NM, but spontaneous cases in dogs have not been genetically characterized. We identified a novel recessive myopathy in a family of line-bred American bulldogs (ABDs); rod bodies in muscle biopsies established this as NM. Using SNP profiles from the nuclear family, we evaluated inheritance patterns at candidate loci and prioritized TNNT1 and NEB for further investigation. Whole exome sequencing of the dam, two affected littermates, and an unaffected littermate revealed a nonsense mutation in NEB (g.52734272 C>A, S8042X). Whole tissue gel electrophoresis and western blots confirmed a lack of full-length NEB in affected tissues, suggesting nonsense-mediated decay. The pathogenic variant was absent from 120 dogs of 24 other breeds and 100 unrelated ABDs, suggesting that it occurred recently and may be private to the family. This study presents the first molecularly characterized large animal model of NM, which could provide new opportunities for therapeutic approaches.     

DNA binding studies of a new dicationic porphyrin. Insights into interligand interactions

Cationic porphyrins have an affinity for DNA and potential for applications in the fields of photodynamic therapy and cellular imaging. This report describes a new dicationic porphyrin, 5,15-dimethyl-10,20-di(N-methylpyridinium-4-yl)porphyrin, abbreviated H2tMe2D4. Although tetrasubstituted, H2tMe2D4 presents modest steric requirements and forms in reasonable yield by a "2+2" synthetic method. Accordingly, studies of the zinc(II)- and copper(II)-containing derivatives, Zn(tMe2D4) and Cu(tMe2D4), have also been possible. Methods used to characterize DNA-binding motifs include absorption, emission, linear, and circular dichroism spectroscopies, as well as viscometry. An unusually detailed picture of porphyrin uptake emerges. As the ratio of DNA to porphyrin increases during a typical titration, H2tMe2D4 or Cu(tMe2D4) initially aggregates on the host and then shifts to intercalative binding at close quarters before finally dispersing into non-interacting intercalation sites of the host. Emission studies of the copper(II) porphyrin have been very valuable. The existence of a measurable signal is diagnostic of intercalative binding, and the saturation behavior establishes that internalization typically monopolizes approximately three base pairs. In the moderate loading regime, emission data are most telling because dipole-dipole interactions between near-neighbor porphyrins tend to confuse other spectroscopic assays. The third ligand, Zn(tMe2D4), behaves differently in that the uptake is a strictly cooperative process. The mode of binding also varies with the base content of the DNA host. When the DNA is rich in A=T base pairs, the porphyrin remains five-coordinate and binds externally; however, Zn(tMe2D4) loses its axial ligand and binds by intercalation if the host contains only G[triple bond]C base pairs.     

Ontogeny and stability of separation and threat-induced defensive behaviors in rhesus monkeys during the first year of life

Twenty-six rhesus monkeys were tested repeatedly at 4, 8, and 12 months of age to characterize the expression and development of their defensive responses induced by separation from their mothers and exposure to a potential threat. Results demonstrated that by 4 months of age infant monkeys engaged in adult-like context-dependent responses and adaptively regulated these responses in relation to the changing context. When separated from their mothers and alone, infants at 4 months of age were active and emitted frequent coo vocalizations. However, when exposed to the profile of a human face, infants responded by becoming behaviorally inhibited and freezing. At 8 months of age, a dramatic reduction in infants' separation-induced coos was observed, whereas their duration of threat-induced freezing remained unchanged. At 12 months of age, a further decrease in cooing occurred, while freezing duration was maintained. No sex differences were found in the expression of these behaviors or their developmental patterns. Individual differences in separation-induced cooing and threat-induced freezing were apparent and remained stable over the three ages studied. However, within animals no relation was found between individual differences in cooing and freezing. These data demonstrate important differences in the developmental patterns for the expression of cooing and freezing over the first year of life. Marked individual differences in separation-induced cooing and threat-induced freezing were apparent and remained stable from 4–12 months of age. The data support the hypothesis that these different defensive responses reflect different adaptive responses that likely have different underlying mechanisms. The similarities between these defensive responses in rhesus monkeys and humans suggests that understanding the factors that promote the development of individual differences in monkeys will illuminate important factors that promote individual differences in humans. Am. J. Primatol. 44:125–135, 1998. ©1998 Wiley-Liss, Inc.     

Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo

Protein synthesis is tightly controlled by assembly of an intricate ribonucleoprotein complex at the m⁷GTP-cap on eukaryotic mRNAs. Ensuing linear scanning of the 5′ untranslated region (UTR) is believed to transfer the preinitiation complex to the initiation codon. Eukaryotic mRNAs are characterized by significant 5′ UTR heterogeneity, raising the possibility of differential control of translation initiation rate at individual mRNAs. Curiously, many mRNAs with unconventional, highly structured 5′ UTRs encode proteins with central biological roles in growth control, metabolism, or stress response. The 5′ UTRs of such mRNAs may influence protein synthesis rate in multiple ways, but most significantly they have been implicated in mediating alternative means of translation initiation. Cap-independent initiation bypasses strict control over the formation of initiation intermediates at the m⁷GTP cap. However, the molecular mechanisms that favor alternative means of ribosome recruitment are not understood. Here we provide evidence that eukaryotic initiation factor (eIF) 4G controls cap-independent translation initiation at the c-myc and vascular endothelial growth factor (VEGF) 5′ UTRs in vivo. Cap-independent translation was investigated in tetracycline-inducible cell lines expressing either full-length eIF4G or a C-terminal fragment (Ct) lacking interaction with eIF4E and poly(A) binding protein. Expression of Ct, but not intact eIF4G, potently stimulated cap-independent initiation at the c-myc/VEGF 5′ UTRs. In vitro RNA-binding assays suggest that stimulation of cap-independent translation initiation by Ct is due to direct association with the c-myc/VEGF 5′ UTR, enabling 43S preinitiation complex recruitment. Our work demonstrates that variant translation initiation factors enable unconventional translation initiation at mRNA subsets with distinct structural features.