Determination of glutathione in lymphocytes and possible association of redox state and proliferative capacity of lymphocytes.

The glutathione (GSH) content of mouse T- and B-cells was determined and compared with the GSH content of human peripheral blood lymphocytes and human erythrocytes. Owing to the difficulty of obtaining large numbers of purified lymphocytes, a technique was developed to measure picomolar quantities of GSH. By this technique, mouse T- and B-cells, as well as mouse peripheral-blood lymphocytes, were found to contain approx. 30% of the GSH found in human peripheral-blood lymphocytes. The concanavalin A response of human peripheral-blood lymphocytes and human spleen cells was insensitive to 2-mercaptoethanol as well as to culture in 17% O2, whereas mouse lymphocyte responses were altered by 2-mercaptoethanol and inhibited by 17% O2. The capacity of human peripheral-blood lymphocytes, human erythrocytes, mouse T-cells and mouse B-cells to regenerate GSH stores after chemical oxidation by diamide was tested, and it was found that mouse cells were less capable of regenerating GSH than human erythrocytes or human peripheral-blood lymphocytes. In addition, the latter lymphocytes were less sensitive to oxidation of GSH and to inhibition of proliferation by diamide.     

Expression, purification, and characterization of the cytoplasmic domain of the human IGF-1 receptor using a baculovirus expression system.

The cytoplasmatic domain of the beta-subunit of the human IGF-1 receptor (residues 929-1337) has been overexpressed in insect cells using the baculovirus expression system. Synthesis of the soluble protein (IGFK, M(r) 46 kDa) in Spodoptera frugiperda (Sf9) cells was detected 24 h after infection and maximal accumulation was achieved 40-48 h postinfection. Rapid purification to near homogeneity (>/=95% pure protein) was accomplished by sequential chromatography on Resource-Q and phenyl-Sepharose with a specific activity of 142 nmol/min/mg using poly[Glu:Tyr] as substrate. The purified IGFK showed a preference for Mn(2+) ions and a linear incorporation of (32)P from [gamma-(32)P]ATP over a 20-fold dilution of the protein and was stimulated 20-fold by the polycation poly-L-lysine. Interestingly, the kinase autophosphorylated on tyrosine and serine residues. In contrast, a kinase-negative mutant, IGFK-K1003A, did not undergo phosphorylation on tyrosine or serine residues, respectively, suggesting that IGF-1 receptor kinase is a dual specific kinase.     

Modulation of T-cell functions: I. Effect of 2-mercaptoethanol and macrophages on T-cell proliferation

The in vitro effects of 2-mercaptoethanol (2-ME), macrophages (MØ), and concanavalin A (Con A) on the proliferation of normal spleen cells (NSC), MØ-depleted spleen cells (DSC), T cells, T-cell subpopulations, and B cells were assessed by [³H]thymidine incorporation. 2-ME alone was consistently shown not to be mitogenic for purified T cells; however, 2-ME enhanced the early (Days 1 and 2) Con A (2 μg/ml)-induced response of NSC, DSC, and T-cell preparations, but depressed the late response (Days 4 and 5). 2-ME alone was mitogenic for purified B-cells, as reported previously; and the 2-ME-induced B-cell response was inhibited by Con A. Preincubation of T cells with 2-ME was sufficient for enhanced Con A responsiveness; however, if 2-ME was added 24 hr after the initiation of culture, no alteration of the Con A-induced response was observed. Ly-2,3⁺ T cells were unresponsive to Con A (0.3–20 μg/ml), but the addition of 2-ME or peritoneal cells enhanced the Con A responsiveness of Ly-2,3⁺ T cells over 200-fold. Ly-1⁺ T cells responded with a similar doseresponse and kinetic profile as unselected T cells. Although Ly-1⁺ T cells responded to Con A, unlike Ly-2, 3⁺ T cells, extensive removal of MØ significantly reduced the Con A-induced responsiveness of the Ly-1⁺ T cells. The reactivities of Ly-1⁺ and Ly-2,3⁺ DSC could be reconstituted by the addition of MØ or 2-ME; however, the kinetic response of Ly-1⁺ T cells peaked on Day 2–3, and Ly-2,3⁺ T cells had a delayed response which peaked on Day 4–5. The results indicated that (i) 2-ME and/or MØ accelerate the response kinetics of T-cells to Con A; (ii) T-cell subpopulations have differential requirements for MØ and/or 2-ME in the response to Con A; (iii) T-cell subpopulations exhibit differential dose responsiveness to Con A; and (iiii) 2-ME alters Con A responsiveness by a direct effect on T cells.     

Post-translocation dynamics of black-tailed prairie dogs (Cynomys ludovicianus): A successful conservation and human–wildlife conflict mitigation tool

Prairie dogs have declined by 98% throughout their range in the grasslands of North America. Translocations have been used as a conservation tool to reestablish colonies of this keystone species and to mitigate human–wildlife conflict. Understanding the behavioral responses of prairie dogs to translocation is of utmost importance to enhance the persistence of the species and for species that depend on them, including the critically endangered black-footed ferret. In 2017 and 2018, we translocated 658 black-tailed prairie dogs on the Lower Brule Indian Reservation in central South Dakota, USA, a black-footed ferret recovery site. Here, we describe and evaluate the effectiveness of translocating prairie dogs into augered burrows and soft-released within presumed coteries to reestablish colonies in previously occupied habitat. We released prairie dogs implanted with passive integrated transponders (PIT tags) and conducted recapture events approximately 1-month and 1-year post-release. We hypothesized that these methods would result in a successful translocation and that prairie dogs released as coteries would remain close to where they were released because of their highly social structure. In support of these methods leading to a successful translocation, 69% of marked individuals was captured 1-month post-release, and 39% was captured 1-year post-release. Furthermore, considerable recruitment was observed with 495 unmarked juveniles captured during the 1-year post-release trapping event, and the reestablished colony had more than doubled in the area by 2021. Contrary to our hypothesis, yet to our knowledge a novel finding, there was greater initial movement within the colony 1-month post-release than expected based on recapture locations compared with the published average territory size; however, 1 year after release, most recaptured individuals were captured within the expected territory size when compared to capture locations 1-month post-release. This research demonstrates that while translocating prairie dogs may be socially disruptive initially, it is an important conservation tool.     

Mechanism of triangularis sterni shortening during expiration in dogs

The triangularis sterni in the dog shortens during expiration below its in situ relaxation length (Lr) (J. Appl. Physiol. 61: 539-544, 1986). To assess the mechanism of this expiratory muscle shortening, we have measured the electromyogram and the respiratory changes in length of the canine triangularis sterni in the third and fourth right intercostal space, first before and then after selective denervation. Eleven anesthetized, spontaneously breathing animals were studied in the supine posture; five of them were also studied during postural changes from supine to head-up. Before denervation, the muscle in the supine animals shortened by -12.84 +/- 1.81% Lr. With selective denervation, the amount of expiratory muscle shortening was reduced to only -2.54 +/- 0.71% Lr (P less than 0.001). Similarly, a change from the supine to the head-up posture before denervation promoted an increase in expiratory muscle shortening from -13.58 +/- 3.62 to -21.17 +/- 4.04% Lr (P less than 0.005), but the denervation abolished this increase. Denervating the triangularis sterni, however, did not affect expiratory activation of the internal intercostals. These results demonstrate that the expiratory contraction of the canine triangularis sterni is agonistic in nature, and they suggest that this contraction is responsible for most of the active caudal displacement of the ribs in the upper half of the rib cage.     

Aging modulated by the Drosophila insulin receptor through distinct structure-defined mechanisms

Mutations of the Drosophila melanogaster insulin/IGF signaling system slow aging, while also affecting growth and reproduction. To understand this pleiotropy, we produced an allelic series of single codon substitutions in the Drosophila insulin receptor, InR. We generated InR substitutions using homologous recombination and related each to emerging models of receptor tyrosine kinase structure and function. Three mutations when combined as trans-heterozygotes extended lifespan while retarding growth and fecundity. These genotypes reduced insulin-stimulated Akt phosphorylation, suggesting they impede kinase catalytic domain function. Among these genotypes, longevity was negatively correlated with egg production, consistent with life-history trade-off theory. In contrast, one mutation (InR³⁵³) was located in the kinase insert domain, a poorly characterized element found in all receptor tyrosine kinases. Remarkably, wild-type heterozygotes with InR³⁵³ robustly extended lifespan without affecting growth or reproduction and retained capacity to fully phosphorylate Akt. The Drosophila insulin receptor kinase insert domain contains a previously unrecognized SH2 binding motif. We propose the kinase insert domain interacts with SH2-associated adapter proteins to affect aging through mechanisms that retain insulin sensitivity and are independent of reproduction.