The energy-linked conformational equilibrium in chloroplast ATPase

1. The capacity of chloroplast ATPase to drive energy-requiring processes increases with, but the maximal ΔpH maintained by ATPase is independent of the degree of modification by dithioerythritol.

2. After modification by dithioerythritol, the ATPase remains subject to an energy-linked equilibrium between active and inactive ATPase conformations. This results in an energetic threshold, the magnitude of which is independent of the degree of modification by dithioerythritol or the presence of P_i and/or ADP, but increases with medium pH.     

Sphingolipid signaling in gonadal development and function

Sphingolipid second messengers, such as ceramide and sphingosine-1-phosphate, signal proliferation, differentiation and death in mammalian cells. The object of this article is to highlight the potential impact of this new information on the study of female and male gonadal development and function. Since the generation of competent gametes by both sexes is precisely regulated by maturational (meiotic) and apoptotic (quality-control) checkpoints, it is proposed that lipid signaling molecules serve as important contributors to the regulation of gametogenesis. The function of sphingolipid molecules in mediating stress- or damage-induced apoptosis in the germ line, an event most-likely associated with impaired gonadal function and infertility, is also discussed. Collectively, these areas represent exciting research directions that may ultimately lead to the development of new therapeutics to coordinate and control fertility in males and females.     

Functional dissection of CCR5 coreceptor function through the use of CD4-independent simian immunodeficiency virus strains

With rare exceptions, all simian immunodeficiency virus (SIV) strains can use CCR5 as a coreceptor along with CD4 for viral infection. In addition, many SIV strains are capable of using CCR5 as a primary receptor to infect CD4-negative cells such as rhesus brain capillary endothelial cells. By using coupled fluorescence-activated cell sorter (FACS) and infection assays, we found that even very low levels of CCR5 expression could support CD4-independent virus infection. CD4-independent viruses represent valuable tools for finely dissecting interactions between Env and CCR5 which may otherwise be masked due to the stabilization of these contacts by Env-CD4 binding. Based on the ability of SIV Env to bind to and mediate infection of cells expressing CCR5 chimeras and mutants, we identified the N terminus of CCR5 as a critical domain for direct Env binding and for supporting CD4-independent virus infection. However, the activity of N-terminal domain CCR5 mutants could be rescued by the presence of CD4, indicating that other regions of CCR5 are important for post-binding events that lead to viral entry. Rhesus CCR5 supported CD4-independent infection and direct Env binding more efficiently than did human CCR5 due to a single amino acid difference in the N terminus. Interestingly, uncleaved, oligomeric SIV Env protein bound to both CD4 and CCR5 less efficiently than did monomeric gp120. Finally, several mutations present in chronically infected monkey populations are shown to decrease the ability of CCR5 to serve as a primary viral receptor for the SIV isolates examined.     

Death by design: apoptosis, necrosis and autophagy

Apoptosis is the principal mechanism by which cells are physiologically eliminated in metazoan organisms. During apoptotic death, cells are neatly carved up by caspases and packaged into apoptotic bodies as a mechanism to avoid immune activation. Recently, necrosis, once thought of as simply a passive, unorganized way to die, has emerged as an alternate form of programmed cell death whose activation might have important biological consequences, including the induction of an inflammatory response. Autophagy has also been suggested as a possible mechanism for non-apoptotic death despite evidence from many species that autophagy represents a survival strategy in times of stress. Recent advances have helped to define the function of and mechanism for programmed necrosis and the role of autophagy in cell survival and suicide.     

Role of apoptosis in functional luteolysis in the pregnant rabbit corpus luteum: Evidence of a role for placental-derived factors in promoting luteal cell survival

Corpora lutea (CL) were isolated from one rabbit ovary on days 4, 8, 16 (peak luteal function), 28 (functional regression) and 30 of pregnancy and processed for biochemical analysis of DNA integrity. Analysis of DNA integrity revealed the presence of oligonucleosomal fragments in day 28 and day 30 CL but not in day 16 CL. The extent of low molecular weight (<15 kb) DNA labeling was 6.6 +/- 0.84 fold higher in day 30 as compared to day 16 CL (mean +/- SEM; n = 4, P < 0.01). In a second series of experiments, healthy CL collected from day 16 pregnant rabbits were incubated for 2 h in the absence or presence of 250 microg/ml of placental extract (PE) obtained from day 16 and/or day 30 placentas. Analysis of DNA integrity revealed that extensive apoptosis occurred in CL incubated in medium alone and in medium containing day 30 PE. In contrast, day 16 PE significantly suppressed apoptosis vs control (70 +/- 4%). In a third series of experiments, expression of mRNA for bcl-x and bax was measured by Northern analysis of CL treated without and with day 16 PE using cRNA probes for bcl-x and bax developed in our laboratory by RT-PCR. Treatment with PE significantly reduced bax mRNA levels but did not change bcl-x mRNA levels. These studies provide evidence that functional luteolysis in the pregnant rabbit CL is correlated with the occurrence of apoptosis. The data suggest that a factor(s) derived from the placenta may be responsible for the prolongation of CL life span during pregnancy by its ability to alter the bax:bcl -x rheostat and suppress apoptosis.     

Multi-modality imaging identifies key times for annexin V imaging as an early predictor of therapeutic outcome

Radiolabeled annexin V may provide an early indication of the success or failure of anticancer therapy on a patient-by-patient basis as an in vivo marker of tumor cell killing. An important question that remains is when, after initiation of treatment, should annexin V imaging be performed. To address this issue, we obtained simultaneous in vivo measurements of tumor burden and uptake of radiolabeled annexin V in the syngeneic orthotopic murine BCL1 lymphoma model using in vivo bioluminescence imaging (BLI) and small animal single-photon emission computed tomography (SPECT). BCL1 cells labeled for fluorescence and bioluminescence assays (BCL1-gfp/luc) were injected into mice at a dose that leads to progressive disease within two to three weeks. Tumor response was followed by BLI and SPECT before and after treatment with a single dose of 10 mg/kg doxorubicin. Biodistribution analyses revealed a biphasic increase of annexin V uptake within the tumor-bearing tissues of mice. An early peak occurring before actual tumor cells loss was observed between 1 and 5 hr after treatment, and a second longer sustained rise from 9 to 24 hr after therapy, which heralds the onset of tumor cell loss as confirmed by BLI. Multimodality imaging revealed the temporal patterns of tumor cell loss and annexin V uptake revealing a better understanding of the timing of radiolabeled annexin V uptake for its development as a marker of therapeutic efficacy.     

CD4+CD25+ regulatory T cells preserve graft-versus-tumor activity while inhibiting graft-versus-host disease after bone marrow transplantation

Mature donor T cells cause graft-versus-host disease (GVHD), but they are also the main mediators of the beneficial graft-versus-tumor (GVT) activity of allogeneic bone marrow transplantation. Suppression of GVHD with maintenance of GVT activity is a desirable outcome for clinical transplantation. We have previously shown that donor-derived CD4+CD25+ regulatory T cells inhibit lethal GVHD after allogeneic bone marrow transplantation across major histocompatibility complex (MHC) class I and II barriers in mice. Here we demonstrate that in host mice with leukemia and lymphoma, CD4+CD25+ regulatory T cells suppress the early expansion of alloreactive donor T cells, their interleukin-2-receptor (IL-2R) alpha-chain expression and their capacity to induce GVHD without abrogating their GVT effector function, mediated primarily by the perforin lysis pathway. Thus, CD4+CD25+ T cells are potent regulatory cells that can separate GVHD from GVT activity mediated by conventional donor T cells.     

Non-coordinate regulation of endogenous epithelial sodium channel (ENaC) subunit expression at the apical membrane of A6 cells in response to various transporting conditions

In many epithelial tissues in the body (e.g. kidney distal nephron, colon, airways) the rate of Na(+) reabsorption is governed by the activity of the epithelial Na(+) channel (ENaC). ENaC activity in turn is regulated by a number of factors including hormones, physiological conditions, and other ion channels. To begin to understand the mechanisms by which ENaC is regulated, we have examined the trafficking and turnover of ENaC subunits in A6 cells, a polarized, hormonally responsive Xenopus kidney cell line. As previously observed by others, the half-life of newly synthesized ENaC subunits was universally short ( approximately 2 h). However, the half-lives of alpha- and gamma-ENaC subunits that reached the apical cell surface were considerably longer (t(12) > 24 h), whereas intriguingly, the half-life of cell surface beta-ENaC was only approximately 6 h. We then examined the effects of various modulators of sodium transport on cell surface levels of individual ENaC subunits. Up-regulation of ENaC-mediated sodium conductance by overnight treatment with aldosterone or by short term incubation with vasopressin dramatically increased cell surface levels of beta-ENaC without affecting alpha- or gamma-ENaC levels. Conversely, treatment with brefeldin A selectively decreased the amount of beta-ENaC at the apical membrane. Short term treatment with aldosterone or insulin had no effect on cell surface amounts of any subunits. Subcellular fractionation revealed a selective loss of beta-ENaC from early endosomal pools in response to vasopressin. Our data suggest the possibility that trafficking and turnover of individual ENaC subunits at the apical membrane of A6 cells is non-coordinately regulated. The selective trafficking of beta-ENaC may provide a mechanism for regulating sodium conductance in response to physiological stimuli.