Social stress results in altered glucocorticoid regulation and shorter survival in simian acquired immune deficiency syndrome

From early in the AIDS epidemic, psychosocial stressors have been proposed as contributors to the variation in disease course. To test this hypothesis, rhesus macaques were assigned to stable or unstable social conditions and were inoculated with the simian immunodeficiency virus. Animals in the unstable condition displayed more agonism and less affiliation, shorter survival, and lower basal concentrations of plasma cortisol compared with stable animals. Early after inoculation, but before the emergence of group differences in cortisol levels, animals receiving social threats had higher concentrations of simian immunodeficiency virus RNA in plasma, and those engaging in affiliation had lower concentrations. The results indicate that social factors can have a significant impact on the course of immunodeficiency disease. Socially induced changes in pituitary–adrenal hormones may be one mechanism mediating this relationship.

Recessive and dominant mutations in the ethylene biosynthetic gene ACS5 of Arabidopsis confer cytokinin insensitivity and ethylene overproduction, respectively

We identified a set of cytokinin-insensitive mutants by using a screen based on the ethylene-mediated triple response observed after treatment with low levels of cytokinins. One group of these mutants disrupts ACS5, a member of the Arabidopsis gene family that encodes 1-aminocyclopropane-1-carboxylate synthase, the first enzyme in ethylene biosynthesis. The ACS5 isoform is mainly responsible for the sustained rise in ethylene biosynthesis observed in response to low levels of cytokinin and appears to be regulated primarily by a posttranscriptional mechanism. Furthermore, the dominant ethylene-overproducing mutant eto2 was found to be the result of an alteration of the carboxy terminus of ACS5, suggesting that this domain acts as a negative regulator of ACS5 function.

Linking topography of its potential surface with the dynamics of folding of a protein model

The “3-color, 46-bead” model of a folding polypeptide is the vehicle for adapting to proteins a mode of analysis used heretofore for atomic clusters, to relate the topography of the potential surface to the dynamics that lead to formation of selected structures. The analysis is based on sequences of stationary points—successive minima, joined by saddles—that rise monotonically in energy from basin bottoms. Like structure-seeking clusters, the potential surface of the model studied here is staircase-like, rather than sawtooth-like, with highly collective motions required for passage from one minimum to the next. The surface has several deep basins whose minima correspond to very similar structures, but which are separated by high energy barriers.

A human intermediate conductance calcium-activated potassium channel

An intermediate conductance calcium-activated potassium channel, hIK1, was cloned from human pancreas. The predicted amino acid sequence is related to, but distinct from, the small conductance calcium-activated potassium channel subfamily, which is ≈50% conserved. hIK1 mRNA was detected in peripheral tissues but not in brain. Expression of hIK1 in Xenopus oocytes gave rise to inwardly rectifying potassium currents, which were activated by submicromolar concentrations of intracellular calcium (K0.5 = 0.3 μM). Although the K0.5 for calcium was similar to that of small conductance calcium-activated potassium channels, the slope factor derived from the Hill equation was significantly reduced (1.7 vs. 3.5). Single-channel current amplitudes reflected the macroscopic inward rectification and revealed a conductance level of 39 pS in the inward direction. hIK1 currents were reversibly blocked by charybdotoxin (Ki = 2.5 nM) and clotrimazole (Ki = 24.8 nM) but were minimally affected by apamin (100 nM), iberiotoxin (50 nM), or ketoconazole (10 μM). These biophysical and pharmacological properties are consistent with native intermediate conductance calcium-activated potassium channels, including the erythrocyte Gardos channel.

Gi regulation of secretory vesicle swelling examined by atomic force microscopy

In the last decade, several monomeric and heterotrimeric guanine nucleotide binding proteins have been identified to associate with secretory vesicles and to be implicated in exocytosis. Vesicle volume also has been proposed to play a regulatory role in secretory vesicle fusion at the plasma membrane. However, the molecular mechanism of function of the guanine nucleotide binding proteins and of the regulation of secretory vesicle volume in the exocytotic process remains unclear. In this study, we report association of the secretory vesicle membrane with the α subunit of a heterotrimeric GTP binding protein Gαi3 and implicate its involvement in vesicle swelling. Using an atomic force microscope in combination with confocal microscopy, we were able to study the dynamics of isolated zymogen granules, the secretory vesicles in exocrine pancreas. Exposure of zymogen granules to GTP resulted in a 15–25% increase in vesicle height as measured by the atomic force microscope and a similar increase in vesicle diameter as determined by confocal microscopy. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and KCl-sensitive. Ca2+ had no effect on zymogen granule size. Taken together, the results indicate that Gαi3 protein localized in the secretory vesicle membrane mediates vesicle swelling, a potentially important prerequisite for vesicle fusion at the cell plasma membrane.

NF-κB activation provides the potential link between inflammation and hyperplasia in the arthritic joint

The transcription factor NF-κB is a pivotal regulator of inflammatory responses. While the activation of NF-κB in the arthritic joint has been associated with rheumatoid arthritis (RA), its significance is poorly understood. Here, we examine the role of NF-κB in animal models of RA. We demonstrate that in vitro, NF-κB controlled expression of numerous inflammatory molecules in synoviocytes and protected cells against tumor necrosis factor α (TNFα) and Fas ligand (FasL) cytotoxicity. Similar to that observed in human RA, NF-κB was found to be activated in the synovium of rats with streptococcal cell wall (SCW)-induced arthritis. In vivo suppression of NF-κB by either proteasomal inhibitors or intraarticular adenoviral gene transfer of super-repressor IκBα profoundly enhanced apoptosis in the synovium of rats with SCW- and pristane-induced arthritis. This indicated that the activation of NF-κB protected the cells in the synovium against apoptosis and thus provided the potential link between inflammation and hyperplasia. Intraarticular administration of NF-kB decoys prevented the recurrence of SCW arthritis in treated joints. Unexpectedly, the severity of arthritis also was inhibited significantly in the contralateral, untreated joints, indicating beneficial systemic effects of local suppression of NF-κB. These results establish a mechanism regulating apoptosis in the arthritic joint and indicate the feasibility of therapeutic approaches to RA based on the specific suppression of NF-κB.

Transglutaminase-catalyzed inactivation of glyceraldehyde 3-phosphate dehydrogenase and α-ketoglutarate dehydrogenase complex by polyglutamine domains of pathological length

Several adult-onset neurodegenerative diseases are caused by genes with expanded CAG triplet repeats within their coding regions and extended polyglutamine (Qn) domains within the expressed proteins. Generally, in clinically affected individuals n ≥ 40. Glyceraldehyde 3-phosphate dehydrogenase binds tightly to four Qn disease proteins, but the significance of this interaction is unknown. We now report that purified glyceraldehyde 3-phosphate dehydrogenase is inactivated by tissue transglutaminase in the presence of glutathione S-transferase constructs containing a Qn domain of pathological length (n = 62 or 81). The dehydrogenase is less strongly inhibited by tissue transglutaminase in the presence of constructs containing shorter Qn domains (n = 0 or 10). Purified α-ketoglutarate dehydrogenase complex also is inactivated by tissue transglutaminase plus glutathione S-transferase constructs containing pathological-length Qn domains (n = 62 or 81). The results suggest that tissue transglutaminase-catalyzed covalent linkages involving the larger poly-Q domains may disrupt cerebral energy metabolism in CAG/Qn expansion diseases.

A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5

HIV-1 entry into CD4+ cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. One such inhibitor has been described for CCR5: the TAK-779 molecule. To facilitate the further development of entry inhibitors as antiviral drugs, we have explored how TAK-779 acts to prevent HIV-1 infection, and we have mapped its site of interaction with CCR5. We find that TAK-779 inhibits HIV-1 replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gp120 with CCR5. We could identify no amino acid substitutions within the extracellular domain of CCR5 that affected the antiviral action of TAK-779. However, alanine scanning mutagenesis of the transmembrane domains revealed that the binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7.

p53 regulates a G2 checkpoint through cyclin B1

The p53 tumor suppressor controls multiple cell cycle checkpoints regulating the mammalian response to DNA damage. To identify the mechanism by which p53 regulates G2, we have derived a human ovarian cell that undergoes p53-dependent G2 arrest at 32°C. We have found that p53 prevents G2/M transition by decreasing intracellular levels of cyclin B1 protein and attenuating the activity of the cyclin B1 promoter. Cyclin B1 is the regulatory subunit of the cdc2 kinase and is a protein required for mitotic initiation. The ability of p53 to control mitotic initiation by regulating intracellular cyclin B1 levels suggests that the cyclin B-dependent G2 checkpoint has a role in preventing neoplastic transformation.

Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells

The pancreatic acinar cell produces powerful digestive enzymes packaged in zymogen granules in the apical pole. Ca2+ signals elicited by acetylcholine or cholecystokinin (CCK) initiate enzyme secretion by exocytosis through the apical membrane. Intracellular enzyme activation is normally kept to a minimum, but in the often-fatal human disease acute pancreatitis, autodigestion occurs. How the enzymes become inappropriately activated is unknown. We monitored the cytosolic Ca2+ concentration ([Ca2+]i), intracellular trypsin activation, and its localization in isolated living cells with specific fluorescent probes and studied intracellular vacuole formation by electron microscopy as well as quantitative image analysis (light microscopy). A physiological CCK level (10 pM) eliciting regular Ca2+ spiking did not evoke intracellular trypsin activation or vacuole formation. However, stimulation with 10 nM CCK, evoking a sustained rise in [Ca2+]i, induced pronounced trypsin activation and extensive vacuole formation, both localized in the apical pole. Both processes were abolished by preventing abnormal [Ca2+]i elevation, either by preincubation with the specific Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N-N′,N′-tetraacetic acid (BAPTA) or by removal of external Ca2+. CCK hyperstimulation evokes intracellular trypsin activation and vacuole formation in the apical granular pole. Both of these processes are mediated by an abnormal sustained rise in [Ca2+]i.

Water does not flow across the tight junctions of MDCK cell epithelium

Although it has been known for decades that the tight junctions of fluid-transporting epithelia are leaky to ions, it has not been possible to determine directly whether significant transjunctional water movement also occurs. An optical microscopic technique was developed for the direct visualization of the flow velocity profiles within the lateral intercellular spaces of a fluid-absorptive, cultured renal epithelium (MDCK) and used to determine the velocity of the fluid flow across the tight junction. The flow velocity within the lateral intercellular spaces fell to near zero adjacent to the tight junction, showing that significant transjunctional flow did not occur, even when transepithelial fluid movement was augmented by imposition of osmotic gradients.

Segment-specific pattern of sympathetic preganglionic projections in the chicken embryo spinal cord is altered by retinoids

Sympathetic preganglionic neurons exhibit segment-specific projections. Preganglionic neurons located in rostral spinal segments project rostrally within the sympathetic chain, those located in caudal spinal segments project caudally, and those in midthoracic segments project either rostrally or caudally in segmentally graded proportions. Moreover, rostrally and caudally projecting preganglionic neurons are skewed toward the rostral and caudal regions, respectively, of each midthoracic segment. The mechanisms that establish these segment-specific projections are unknown. Here we show that experimental manipulation of retinoid signaling in the chicken embryo alters the segment-specific pattern of sympathetic preganglionic projections and that this effect is mediated by the somitic mesoderm. Application of exogenous retinoic acid to a single rostral thoracic somite decreases the number of rostrally projecting preganglionic neurons at that level. Conversely, disrupting endogenous synthesis of retinoic acid in a single caudal thoracic somite increases the number of rostrally projecting preganglionic neurons at that level. The number of caudally projecting neurons does not change in either case, indicating that the effect is specific for rostrally projecting preganglionic neurons. These results indicate that the sizes of the rostrally and caudally projecting populations may be independently regulated by different factors. Opposing gradients of such factors along the longitudinal axis of the thoracic region of the embryo could be sufficient, in combination, to determine the segment-specific identity of preganglionic projections.

A genome-wide search for chromosomal loci linked to mental health wellness in relatives at high risk for bipolar affective disorder among the Old Order Amish

Bipolar affective disorder (BPAD; manic-depressive illness) is characterized by episodes of mania and/or hypomania interspersed with periods of depression. Compelling evidence supports a significant genetic component in the susceptibility to develop BPAD. To date, however, linkage studies have attempted only to identify chromosomal loci that cause or increase the risk of developing BPAD. To determine whether there could be protective alleles that prevent or reduce the risk of developing BPAD, similar to what is observed in other genetic disorders, we used mental health wellness (absence of any psychiatric disorder) as the phenotype in our genome-wide linkage scan of several large multigeneration Old Order Amish pedigrees exhibiting an extremely high incidence of BPAD. We have found strong evidence for a locus on chromosome 4p at D4S2949 (maximum genehunter-plus nonparametric linkage score = 4.05, P = 5.22 × 10−4; sibpal Pempirical value <3 × 10−5) and suggestive evidence for a locus on chromosome 4q at D4S397 (maximum genehunter-plus nonparametric linkage score = 3.29, P = 2.57 × 10−3; sibpal Pempirical value <1 × 10−3) that are linked to mental health wellness. These findings are consistent with the hypothesis that certain alleles could prevent or modify the clinical manifestations of BPAD and perhaps other related affective disorders.