A DEVD-Inhibited Caspase Other than CPP32 Is Involved in the Commitment of Cerebellar Granule Neurons to Apoptosis Induced by K+ Deprivation

Abstract: Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K⁺ (25 mM KCI) to medium containing lower levels of K⁺ (5 mM KCI). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp·(O-methyl)·fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp·fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10–25 µM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp·chloromethyl ketone (YVAD·cmk), an interleukin-1β converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp·fluoromethyl ketone (DEVD·fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD·cmk provided only modest (<20%) protection and only at the highest concentration (100 µM) tested, suggesting that interleukin-1β converting enzyme and/or closely related caspases were not involved. In comparison, DEVD·fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD·fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K⁺-deprived granule neurons.     

The coreceptor CD2 uses plasma membrane microdomains to transduce signals in T cells

The interaction between a T cell and an antigen-presenting cell (APC) can trigger a signaling response that leads to T cell activation. Prior studies have shown that ligation of the T cell receptor (TCR) triggers a signaling cascade that proceeds through the coalescence of TCR and various signaling molecules (e.g., the kinase Lck and adaptor protein LAT [linker for T cell activation]) into microdomains on the plasma membrane. In this study, we investigated another ligand–receptor interaction (CD58–CD2) that facilities T cell activation using a model system consisting of Jurkat T cells interacting with a planar lipid bilayer that mimics an APC. We show that the binding of CD58 to CD2, in the absence of TCR activation, also induces signaling through the actin-dependent coalescence of signaling molecules (including TCR-ζ chain, Lck, and LAT) into microdomains. When simultaneously activated, TCR and CD2 initially colocalize in small microdomains but then partition into separate zones; this spatial segregation may enable the two receptors to enhance signaling synergistically. Our results show that two structurally distinct receptors both induce a rapid spatial reorganization of molecules in the plasma membrane, suggesting a model for how local increases in the concentration of signaling molecules can trigger T cell signaling.     

Visualization of mitochondrial coupling factor F1(ATPase) by freeze-drying

It is known that the negatively stained preparations of inner mitochondrial membrane display characteristic ∼9 nmF ₁ (ATPase) knobs projecting from the matrix surface. Freeze-etch studies have reported the absence of such knobs from the “etched” surface of the inner mitochondrial membranes. We have demonstrated their presence on the surface of SMP (submitochondrial particles) prepared by freeze-drying for transmission electron microscopy. This identification has been substantiated by comparison with the freeze-dried TU particles (trypsin-urea treated SMP) that are devoid ofF ₁ (ATPase). It has been suggested that a layer of water molecules is strongly adsorbed to the surface of SMP and does not sublime during normal freeze-“etching.”     

Study of uranium level in groundwater of Balod district of Chhattisgarh state,India and assessment of health risk

Uranium contamination in groundwater at Balod district of Chhattisgarh state was measured by laser fluorimetric technique. Most of the samples were found under safe limit for uranium contamination as recommended by WHO and USEPA (<30 µg l^?1) except Deur Tarai village (78.93 µg l^?1).The excess lifetime carcinogenic risk and chemical risk due to ingestion of groundwater were calculated using USEPA recommendations.     

Agronomic and chemical performance of field‐grown tobacco engineered for triterpene and methylated triterpene metabolism

Squalene is a linear intermediate to nearly all classes of triterpenes and sterols and is itself highly valued for its use in wide range of industrial applications. Another unique linear triterpene is botryococcene and its methylated derivatives generated by the alga Botryococcus braunii race B, which are progenitors to fossil fuel deposits. Production of these linear triterpenes was previously engineered into transgenic tobacco by introducing the key steps of triterpene metabolism into the particular subcellular compartments. In this study, the agronomic characteristics (height, biomass accumulation, leaf area), the photosynthetic capacity (photosynthesis rate, conductance, internal CO₂ levels) and triterpene content of select lines grown under field conditions were evaluated for three consecutive growing seasons. We observed that transgenic lines targeting enzymes to the chloroplasts accumulated 50–150 times more squalene than the lines targeting the enzymes to the cytoplasm, without compromising growth or photosynthesis. We also found that the transgenic lines directing botryococcene metabolism to the chloroplast accumulated 10‐ to 33‐fold greater levels than the lines where the same enzymes were targeted to in the cytoplasm. However, growth of these high botryococcene accumulators was highly compromised, yet their photosynthesis rates remained unaffected. In addition, in the transgenic lines targeting a triterpene methyltransferase (TMT) to the chloroplasts of high squalene accumulators, 55%–65% of total squalene was methylated, whereas in the lines expressing a TMT in the cytoplasm, only 6%–13% of squalene was methylated. The growth of these methylated triterpene‐accumulating lines was more compromised than that of nonmethylated squalene lines.     

Glucose metabolism at high density growth of E. coli B and E. coli K: Differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and northern blot analyses

The original article to which this Erratum refers was published in Biotechnol Bioeng 2005;90:805–820     

Structural and thermodynamic consequences of 1-(4-chlorophenyl)imidazole binding to cytochrome P450 2B4

The crystal structure of P450 2B4 bound with 1-(4-chlorophenyl)imidazole (1-CPI) has been determined to delineate the structural basis for the observed differences in binding affinity and thermodynamics relative to 4-(4-chlorophenyl)imidazole (4-CPI). Compared with the previously reported 4-CPI complex, there is a shift in the 1-CPI complex of the protein backbone in helices F and I, repositioning the side chains of Phe-206, Phe-297, and Glu-301, and leading to significant reshaping of the active site. Phe-206 and Phe-297 exchange positions, with Phe-206 becoming a ligand-contact residue, while Glu-301, rather than hydrogen bonding to the ligand, flips away from the active site and interacts with His-172. As a result the active site volume expands from 200 A3 in the 4-CPI complex to 280 A3 in the 1-CPI complex. Based on the two structures, it was predicted that a Phe-206-->Ala substitution would alter 1-CPI but not 4-CPI binding. Isothermal titration calorimetry experiments indicated that this substitution had no effect on the thermodynamic signature of 4-CPI binding to 2B4. In contrast, relative to wild-type 1-CPI binding to F206A showed significantly less favorable entropy but more favorable enthalpy. This result is consistent with loss of the aromatic side chain and possible ordering of water molecules, now able to interact with Glu-301 and exposed residues in the I-helix. Hence, thermodynamic measurements support the active site rearrangement observed in the crystal structure of the 1-CPI complex and illustrate the malleability of the active site with the fine-tuning of residue orientations and thermodynamic signatures.     

Two picrotoxin derivatives from Anamirta cocculus

The berries of the plant Anamirta cocculus afforded picrotin, picrotoxinin, methyl picrotoxate and two new sesquiterpene γ-lactones, dihydroxypicrotoxinin and picrotoxic acid, whose structures were determined by spectroscopic methods.     

The Synthetic Tryptanthrin Analogue Suppresses STAT3 Signaling and Induces Caspase Dependent Apoptosis via ERK Up Regulation in Human Leukemia HL-60 Cells

Tryptanthrin is a natural product which has been reported to have several medicinal properties. In this study, we tried to investigate the detailed molecular mechanism of its bromo analogue (TBr), a potent cytotoxic agent in the induction of cancer cell death. It was found that TBr primarily targets STAT3 and ERK signaling during the induction of apoptosis in several human leukemia cell lines. In HL-60 cells, TBr treatment caused early down regulation of p-STAT3 with concomitant up regulation of p-ERK which led to the activation of intrinsic and extrinsic pathways of apoptosis. The mechanism of TBr mediated inhibition of p-STAT3 was found to be due to the activation of ubiquitin dependent degradation of tyrosine 705 and serine 727 p-STAT3. As IL-6 is the main driver of the STAT3 pathway, the effect of TBr on cell death was subdued when treated in the combination with IL-6 in HL60 cells. Interestingly, PD98059 significantly reduced the apoptotic effects of TBr, thus showing the direct involvement of p-ERK in TBr mediated cell death. It was further shown that apoptotic protein Bax silencing in HL-60 cells resists TBr mediated ERK dependent apoptosis. In summary, for the first time we report the mechanism of TBr mediated cell death in human leukemia cell lines by targeting STAT3 and ERK pathways.     

Individual Management of Recurrent Intracranial Aneurysms: The Wuxi Experience

Recurrent intracranial aneurysms can occur after either surgical clipping or endovascular therapy. In this article, we present a consecutive series of 18 patients who underwent individual treatment for recurrent aneurysms after primary coil embolization or surgical clipping. During an 8-year period between May 1997 and December 2005, 18 patients underwent individual treatment for recurrent aneurysms. Clinical data and imaging studies of the patients were analyzed retrospectively. Out of the 18 patients, 13 had recurrent aneurysms located in the anterior circulation, and 5 had aneurysms of the posterior circulation. Treatment consisted of coiling in 16 patients and clipping in two patients. Of the 18 patients, 15 achieved a good or excellent recovery, two were paralyzed, and one died post-treatment. Both the surgical clipping and endovascular embolization for the treatment of recurrent intracranial aneurysms can achieve very good radiological results with low mortality rates. One of the key points for the successful treatment of this kind of lesions is the proper, individual, and interdisciplinary patient selection.