Sequential activation and loss of the pre-B cell Thy-1 gene in T-cell x pre-B cell somatic hybrids

In somatic cell hybrids between the pseudodiploid Thy-1^– Abelson-leukemia-virus-induced pre-B cell lymphoma RAW 253.1 and the Thy-1⁺ T-cell lymphoma, AKR1 (Thy-1⁺), all cells express the Thy-1 allele of the T-cell parent but most hybrid cells do not express the Thy-1 allele of the pre-B cell lymphoma parent. The Thy-1 allele of the pre-B cell parent, however, is spontaneously activated in a minor proportion of hybrid cells. By sorting for cells expressing the Thy-1 allele of the pre-B cell parent, derivative clones in which 100% of cells express both parental Thy-1 alleles can be isolated. Revertants with a phenotype identical with that of the original hybrid cell line can be isolated from these derivatives by sorting for nonexpression of the Thy-1 allele of the pre-B cell parent. These first-generation revertant cell lines have lost one copy of the Thy-1 gene derived from the pre-B cell lymphoma parent. By a further cycle of sorting, derivatives in which 100% of cells express both parental Thy-1 alleles can again be obtained. Second-generation revertants isolated by sorting these Thy-1⁺ hybrid cells for nonexpression of the Thy-1 allele of the pre-B cell parent no longer contain a normal copy of the pre-B cell Thy-1 allele and this surface antigen is no longer expressed by any cells in the population. These results are consistent with a mechanism that sequentially activates each copy of the Thy-1 gene derived from the pre-B cell lymphoma parent. Hybrids between the class D Thy-1 ^– mutant, AKR1(Thy-1^–d), in which the 5 region of the Thy-1 structural gene has been deleted, and RAW 253.1 cannot be activated to express either Thy-1 allele. This result indicates that a sequence upstream of exon 2 of the active Thy-1 allele is critical for the initial activation event.     

Factors affecting the induction of secondary dormancy in lettuce

The relationship between the temperature at which germination of 50% of the seeds is inhibited in the light (GT₅₀ Light) and secondary dormancy was investigated in three cultivars of Lactuca sativa L. Seeds were incubated for varying periods under non-germinating conditions and subsequent germination in response to red light (R) was determined over a wide range of temperatures. Dark incubation at 32 C reduced the GT₅₀ Light of cv. New York but did not affect germination at temperatures below 24 C. Dark, 32 C incubation had no effect on the GT₅₀ Light of cv. Great Lakes. In cv. Grand Rapids, dark incubation at 15, 24, 32, or 35 C initially reduced the GT₅₀ Light. However, longer incubations induced a secondary dormancy, i.e., the seeds became unable to germinate at all temperatures in response to R given after the high temperature incubation. A single exposure to R at the beginning of a 32 C incubation slowed the induction of secondary dormancy. Repeated exposures to R prevented the induction of secondary dormancy, but did not prevent a decline in the GT₅₀ Light. GA₃ mimicked the effect of repeated R.     

Potential benefits of gene editing for the future of poultry farming

The chicken is an exemplar of efficient intensive animal agriculture and provides two valuable food products, chicken meat and eggs. Only aquaculture is better, by efficiency, but poultry is still top, by mass of animal protein produced as food in the global context. However this efficiency and intensive production comes with a number of challenges. Though the genetics of selective breeding have led to dramatic improvements in yield, efficiency and product quality, traits that relate to disease and welfare outcomes have not been so tractable. These two issues are major impacts to the industry in terms of production and in terms of public perception. Both transgenic technology and genome editing have clear potential for impact in these two important areas. The reproductive biology of birds requires techniques very specific to birds to achieve heritable (germline) edited traits. These are quite involved and, even though they are now well-defined and reliable, there is room for improvement and advances can be expected in the future. Currently the key targets for this technology are modifying chicken genes involved in virus-receptor interactions and cellular response involved in infection. For the egg industry the technology is being applied to the issue of sex-selection for layer hens (and the removal of males), removal of allergens from egg white and the tailoring of eggs system to enhance the yield of influenza vaccine doses. Regulation and trading of the animals generated, and resulting food products, will significantly impact the value and future development of genome editing for poultry.

     

ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species

ANG II constricts descending vasa recta (DVR) through Ca(2+) signaling in pericytes. We examined the role of PKC DVR pericytes isolated from the rat renal outer medulla. The PKC blocker staurosporine (10 microM) eliminated ANG II (10 nM)-induced vasoconstriction, inhibited pericyte cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) elevation, and blocked Mn(2+) influx into the cytoplasm. Activation of PKC by either 1,2-dioctanoyl-sn-glycerol (10 microM) or phorbol 12,13-dibutyrate (PDBu; 1 microM) induced both vasoconstriction and pericyte [Ca(2+)](cyt) elevation. Diltiazem (10 microM) blocked the ability of PDBu to increase pericyte [Ca(2+)](cyt) and enhance Mn(2+) influx. Both ANG II- and PDBu-induced PKC stimulated DVR generation of reactive oxygen species (ROS), measured by oxidation of dihydroethidium (DHE). The effect of ANG II was only significant when ANG II AT(2) receptors were blocked with PD-123319 (10 nM). PDBu augmentation of DHE oxidation was blocked by either TEMPOL (1 mM) or diphenylene iodonium (10 microM). We conclude that ANG II and PKC activation increases DVR pericyte [Ca(2+)](cyt), divalent ion conductance into the cytoplasm, and ROS generation.     

Striatal Damage and Oxidative Stress Induced by the Mitochondrial Toxin Malonate Are Reduced in Clorgyline-Treated Rats and MAO-A Deficient Mice

Intrastriatal administration of the succinate dehydrogenase (SDH) inhibitor malonate produces neuronal injury by a "secondary excitotoxic" mechanism involving the generation of reactive oxygen species (ROS). Recent evidence indicates dopamine may contribute to malonate-induced striatal neurodegeneration; infusion of malonate causes a pronounced increase in extracellular dopamine and dopamine deafferentation attenuates malonate toxicity. Inhibition of the catabolic enzyme monoamine oxidase (MAO) also attenuates striatal lesions induced by malonate. In addition to forming 3,4-dihydroxyphenylacetic acid, metabolism of dopamine by MAO generates H2O2, suggesting that dopamine metabolism may be a source of ROS in malonate toxicity. There are two isoforms of MAO, MAO-A and MAO-B. In this study, we have investigated the role of each isozyme in malonate-induced striatal injury using both pharmacological and genetic approaches. In rats treated with either of the specific MAO-A or -B inhibitors, clorgyline or deprenyl, respectively, malonate lesion volumes were reduced by 30% compared to controls. In knock-out mice lacking the MAO-A isoform, malonate-induced lesions were reduced by 50% and protein carbonyls, an index ROS formation, were reduced by 11%, compared to wild-type animals. In contrast, mice deficient in MAO-B showed highly variable susceptibility to malonate toxicity precluding us from determining the precise role of MAO-B in this form of brain damage. These findings indicate that normal levels of MAO-A participate in expression of malonate toxicity by a mechanism involving oxidative stress.     

Development of a polyvalent assay system for lead identification

In an effort to identify new approaches to lead discovery a polyvalent assay was developed to allow identification of weak inhibitors. This approach involves the polyvalent display of a protein binder off a Tenta-gel scaffold and the generation of a polyvalent display of protein by biotinylation followed by complexation with fluorescently labeled streptavidin. Subsequent exposure of the streptavidin complexed protein to Tenta-gel beads with active protein binders results in fluorescent beads, which are easily viewed under a fluorescent microscope.     

Microtubule-dependent movement of late endocytic vesicles in vitro: requirements for Dynein and Kinesin

Our previous studies demonstrated that fluorescent early endocytic vesicles prepared from rat liver after injection of Texas red asialoorosomucoid contain asialoglycoprotein and its receptor and move and undergo fission along microtubules using kinesin I and KIFC2, with Rab4 regulating KIFC2 activity (J. Cell Sci. 116, 2749, 2003). In the current study, procedures to prepare fluorescent late endocytic vesicles were devised. In addition, flow cytometry was utilized to prepare highly purified fluorescent endocytic vesicles, permitting validation of microscopy-based experiments as well as direct biochemical analysis. These studies revealed that late vesicles bound to and moved along microtubules, but in contrast to early vesicles, did not undergo fission. As compared with early vesicles, late vesicles had reduced association with receptor, Rab4, and kinesin I but were highly associated with dynein, Rab7, dynactin, and KIF3A. Dynein and KIF3A antibodies inhibited late vesicle motility, whereas kinesin I and KIFC2 antibodies had no effect. Dynamitin antibodies prevented the association of late vesicles with microtubules. These results indicate that acquisition and exchange of specific motor and regulatory proteins characterizes and may regulate the transition of early to late endocytic vesicles. Flow cytometric purification should ultimately facilitate detailed proteomic analysis and mapping of endocytic vesicle-associated proteins.     

Effect of two and five days of creatine loading on anaerobic working capacity in women

The purpose of this study was to determine the effects of 2 and 5 days of Cr loading on anaerobic working capacity (AWC) using the critical power (CP) test in women. Ten physically active women randomly received 2 treatments separated by a 5 week washout period: (A) 18 g dextrose as placebo (PL) or (B) 5.0 g Cr + 18 g dextrose taken 4 times per day for 5 days. Following a familiarization trial, each subject completed the CP test at baseline and following 2 and 5 days of supplementation. The PL resulted in no significant changes in AWC following supplementation; however, Cr increased AWC by 22.1% after 5 days of loading (p < 0.05). There was a significant main effect for body weight (BW), however, there was no significant increase in BW due to Cr supplementation. These results suggest that Cr supplementation is effective for increasing AWC in women following 5 days of loading without an associated increase in BW.     

Stromal cells provide the matrix for migration of early lymphoid progenitors through the thymic cortex

During steady state lymphopoiesis in the postnatal thymus, migration of precursors outward from the deep cortex toward the capsule is required for normal differentiation. Such migration requires, at a minimum, expression of adhesive receptors on the migrating lymphoid cells, as well as a stable matrix of their ligands persisting throughout the region of migration. In this study, we address the nature of this adhesive matrix. Although some precursor stages bound efficiently to extracellular matrix ligands, a specific requirement for the cell surface ligand VCAM-1 was also found. In situ analysis revealed that early precursors are found in intimate contact with a matrix formed by stromal cells in the cortex, a proportion of which expresses VCAM-1. In vivo administration of an anti-VCAM-1 Ab resulted in decreased thymic size and altered distribution of early precursors within the cortex. These results indicate that precursors migrating outward through the cortex may use a cellular, rather than extracellular, matrix for adhesion, and suggest that the VCAM-1(+) subset of cortical stroma may play a crucial role in supporting the migration of early precursors in the steady state thymus.