BH3‐in‐groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes

Pro‐apoptotic Bax induces mitochondrial outer membrane permeabilization (MOMP) by forming oligomers through a largely undefined process. Using site‐specific disulfide crosslinking, compartment‐specific chemical labeling, and mutational analysis, we found that activated integral membrane Bax proteins form a BH3‐in‐groove dimer interface on the MOM surface similar to that observed in crystals. However, after the α5 helix was released into the MOM, the remaining interface with α2, α3, and α4 helices was rearranged. Another dimer interface was formed inside the MOM by two intersected or parallel α9 helices. Combinations of these interfaces generated oligomers in the MOM. Oligomerization was initiated by BH3‐in‐groove dimerization, without which neither the other dimerizations nor MOMP occurred. In contrast, α9 dimerization occurred downstream and was required for release of large but not small proteins from mitochondria. Moreover, the release of large proteins was facilitated by α9 insertion into the MOM and localization to the pore rim. Therefore, the BH3‐in‐groove dimerization on the MOM nucleates the assembly of an oligomeric Bax pore that is enlarged by α9 dimerization at the rim.     

Tumor necrosis factor alpha stimulates NMDA receptor activity in mouse cortical neurons resulting in ERK-dependent death

Multiple cytokines are secreted in the brain during pro-inflammatory conditions and likely affect neuron survival. Previously, we demonstrated that glutamate and tumor necrosis factor alpha (TNFalpha) kill neurons via activation of the N-methyl-d-aspartate (NMDA) and TNFalpha receptors, respectively. This report continues characterizing the signaling cross-talk pathway initiated during this inflammation-related mechanism of death. Stimulation of mouse cortical neuron cultures with TNFalpha results in a transient increase in NMDA receptor-dependent calcium influx that is additive with NMDA stimulation and inhibited by pre-treatment with the NMDA receptor antagonist, DL-2-amino-5-phosphonovaleric acid, or the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione. Pre-treatment with N-type calcium channel antagonist, omega-conotoxin, or the voltage-gated sodium channel antagonist, tetrodotoxin, also prevents the TNFalpha-stimulated calcium influx. Combined TNFalpha and NMDA stimulation results in a transient increase in activity of extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs). Specific inhibition of ERKs but not JNKs is protective against TNFalpha and NMDA-dependent death. Death is mediated via the low-affinity TNFalpha receptor, TNFRII, as agonist antibodies for TNFRII but not TNFRI stimulate NMDA receptor-dependent calcium influx and death. These data demonstrate how microglial pro-inflammatory secretions including TNFalpha can acutely facilitate glutamate-dependent neuron death.     

Reduced adiposity in ob/ob mice following total body irradiation and bone marrow transplantation

Objective: The objective of this study was to assess long‐term metabolic consequences of total body irradiation (TBI) and bone marrow transplantation. Severe obesity develops due to both hypertrophy and hyperplasia of adipocytes. We hypothesized that TBI would arrest adipose tissue growth and would affect insulin resistance (IR). Research Methods and Procedures: We exposed 2‐month‐old female ob/ob mice to 8 Grays of TBI followed by bone marrow transplantation and tested the animals for body weight (BW) gain, body composition, blood glucose, and insulin sensitivity. Results: Two months after TBI, irradiated mice stopped gaining BW, whereas non‐treated mice continued to grow. At the age of 9.5 months, body mass of irradiated mice was 60.6 ± 1.4 grams, which was only 61% of that in non‐treated ob/ob controls (99.4 ± 1.6 grams). Body composition measurements by DXA showed that decreased BW was primarily due to an impaired fat accumulation. This could not result from the production of leptin by bone marrow‐derived adipocyte progenitors because inhibition of the obese phenotype was identical in recipients of both B6 and ob/ob bone marrow. Inability of the irradiated mice to accumulate fat was associated with hepatomegaly, lower levels of monocyte chemoattractant protein‐1 expression in adipose tissue, and increased IR. Discussion: Our data argue in favor of the hypothesis that inability of adipose tissue to expand may increase IR. This mouse model may be valuable for studies of late‐onset radiation‐induced IR in humans.     

Serotonin in a diencephalic nucleus controlling communication in an electric fish: sexual dimorphism and relationship to indicators of dominance

Serotonin regulates aggressive behavior. The production or release of serotonin is sexually dimorphic and related to social rank in many species. We examined serotonin expression in the central posterior/prepacemaker nucleus (CP/PPn) of the electric fish Apteronotus leptorhynchus. The CP/PPn is a thalamic nucleus that controls agonistic and reproductive electrocommunication signals known as chirps and gradual frequency rises. In parts of the CP/PPn that control chirping, females had more than twice as many serotonergic fibers and terminals as did males. Serotonin immunoreactivity in chirp-controlling areas of the CP/PPn was also negatively correlated with two indicators of dominance: electric organ discharge (EOD) frequency and body mass. Within sexes, the negative correlation between EOD frequency and serotonergic innervation of the PPn was significant in females, but not in males. Females with higher EOD frequencies had less serotonin in the CP/PPn than did females with lower EOD frequencies. Thus, the CP/PPn contained more serotonin in females than in males, and in particular, more serotonin in females with EOD frequencies typical of social subordinates than in females with EOD frequencies typical of social dominants. These results, combined with previous findings that serotonin inhibits chirping and that females chirp much less than males, suggest that serotonin may link sex, social rank, and the production of agonistic communication signals. The relative simplicity of the neural circuits that control the EOD and chirping make the electromotor system well-suited for studying the cellular, physiological, and behavioral mechanisms by which serotonin modulates agonistic communication.     

Role of light and jasmonic acid signaling in regulating foliar phloem cell wall ingrowth development

Phloem cells adjacent to sieve elements can possess wall invaginations. The role of light and jasmonic acid signaling in wall ingrowth development was examined in pea companion cells (CCs), Arabidopsis thaliana phloem parenchyma cells (PCs), and in Senecio vulgaris (with ingrowths in both cell types). Features characterized included wall ingrowths (from electron microscopic images), foliar vein density and photosynthetic capacity. In Arabidopsis, wall ingrowths were bulky compared with finger-like invaginations in pea and S. vulgaris. Relative to low light (LL), wall invagination in both CCs and PCs was greater in high light (HL). Treatment with methyl jasmonate in LL had no effect on CCs, but increased PC wall ingrowths. LL-to-HL transfer resulted in significantly less wall ingrowth in the fad7-1 fad8-1 (jasmonate-deficient) Arabidopsis mutant relative to the wild type. These results suggest that chloroplast oxidative status, via chloroplast-derived jasmonates, may modulate phloem structure and function. While CC wall ingrowths facilitate phloem loading by expanding the membrane area available for active uptake, one can speculate that phloem PC ingrowths may have two potential roles: to increase the efflux of sugars and/or protons into the apoplast to augment phloem loading; and/or to protect the phloem against pathogens and/or insects.     

Detection of early renal transplant rejection by minimally-invasive monitoring of impedance variability

BACKGROUND: Allograft rejection that occurs after renal transplants is identified by surveillance biopsies or by abnormal laboratory and/or hemodynamic data. The latter are insensitive markers of rejection that may not appear until significant histologic damage has already occurred. Therefore, a sensitive and specific non-invasive method of detecting early rejection of transplanted solid organs is needed. METHOD: A single canine renal allograft was implanted followed by bilateral nephrectomy. Bipolar pacing electrodes were implanted at each end of the transplanted kidney. A second set of electrodes was implanted in the liver, which served as a non-rejecting normal organ. Electrodes were connected to an implantable sensor placed in the subcutaneous tissue. Electrical tissue impedance levels were telemetrically downloaded daily. The clinical status of the transplanted organ was monitored by following the blood urea nitrogen and serum creatinine levels, urine output, and clinical appearance. After tissue impedance levels had stabilized, all immunosuppressants were abruptly discontinued. Clinical signs of rejection were then observed after a few days. RESULTS: Rejection was accompanied by changes in electrical impedance of the implanted organ. These changes, when observed, occurred 1-5 days before clinical signs of rejection appeared. CONCLUSION: Analyses of these data suggest that development of a minimally-invasive high-confidence sensor of early rejection of solid organ transplants is feasible.     

Benzothiazole benzimidazole (S)-isothiazolidinone derivatives as protein tyrosine phosphatase-1B inhibitors

Benzothiazole benzimidazole (S)-isothiazolidinone ((S)-IZD) derivatives 5 were discovered through a peptidomimetic modification of the tripeptide (S)-IZD protein tyrosine phosphatase 1B (PTP1B) inhibitor 1. These derivatives are potent, competitive, and reversible inhibitors of PTP1B with improved caco-2 permeability. An X-ray co-crystal structure of inhibitor 5/PTP1B at 2.2A resolution demonstrated that the benzothiazole benzimidazole forms bi-dentate H-bonds to Asp48, and the benzothiazole interacts with the surface of the protein in a solvent exposed region towards the C-site. The design, synthesis, and SAR of this novel series of benzothiazole benzimidazole containing (S)-IZD inhibitors of PTP1B are presented herein.     

Preactivated carboxyl linker for the rapid conjugation of alkylamines to oligonucleotides on solid support

A C10 linker phosphoramidite reagent terminated with a succinimidyl-activated carboxyl group was prepared and used to couple to the 5'-end of an oligonucleotide synthesized on a solid support. The succinimidyl-activated carboxyl functionality can be used for rapid conjugation of amines to oligonucleotides on solid support or it can be hydrolyzed to form a carboxylic acid functionality. The activated linker was successfully used for conjugation of several primary and secondary aliphatic amine derivatives (including biotin and fluorescein cadaverine) onto a solid support-bound 12-mer DNA oligonucleotide at scales ranging from 0.15 to 1.0 micromol. The overall yields of the conjugation products after AMA deprotection and cleavage from the solid support ranged from 43 to 75% of the total oligonucleotide product. This value is significant, as it includes oligonucleotide synthesis, coupling of the linker, and conjugation of the amine. In addition, the entire process of oligonucleotide synthesis, linker coupling, amine conjugation, deprotection, and cleavage of the oligonucleotide from solid support can be accomplished in 1 day.