Cooperative Interception of Neuronal Apoptosis by BCL-2 and BAG-1 Expression: Prevention of Caspase Activation and Reduced Production of Reactive Oxygen Species

Abstract: Neuronally differentiated PC12 cells undergo synchronous apoptosis when deprived of nerve growth factor (NGF). Here we show that NGF withdrawal induces actinomycin D- and cycloheximide-sensitive caspase (ICE-like) activity. The peptide inhibitor of caspase activity, N -acetyl-Asp-Glu-Val-Asp-aldehyde, was more potent than acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone in preventing NGF withdrawal-induced apoptosis, suggesting an important role for caspase-3 (CPP32)-like proteases. We observed a peak of reactive oxygen species (ROS) 6 h after NGF withdrawal. ROS appear to be required for apoptosis, because cell death is prevented by the free radical spin trap, N-tert -butyl-α-phenylnitrone, and the antioxidant, N -acetylcysteine. ROS production was blocked by actinomycin D, cycloheximide, and caspase protease inhibitors, suggesting that ROS generation is downstream of new mRNA and protein synthesis and activation of caspases. Forced expression of either BCL-2 or the BCL-2-binding protein BAG-1 blocked NGF withdrawal-induced apoptosis, activation of caspases, and ROS generation, showing that they function upstream of caspases. Coexpression of BCL-2 and BAG-1 was more protective than expression of either protein alone.     

The CD4-independent tropism of human immunodeficiency virus type 2 involves several regions of the envelope protein and correlates with a reduced activation threshold for envelope-mediated fusion.

Several human immunodeficiency virus type 2 (HIV-2) strains have been shown to infect some CD4-negative cell lines (P. R. Clapham, A. McKnight, and R. A. Weiss, J. Virol. 66:3531-3537, 1992). Using molecular clones of HIV-2 with a CD4-independent tropism, we have identified critical amino acid residues in the envelope protein which are required for CD4-independent infection. Mutations located immediately upstream of a proposed coiled coil domain in the transmembrane protein (A526T or I528M) and flanking the base of the V4 loop (L378F and K403R) are crucial for the CD4-independent phenotype. Of several mutations conferring a positive charge in V1, V2, and V3, only the change in V3 (Q310K) helped to enhance the CD4-independent phenotype but could not mediate it on its own. These mutations reduce the amount of soluble CD4 required to trigger CD4-independent cell-cell fusion, suggesting that they lower the activation threshold for the fusion process. After binding to cell surface-anchored CD4, a CD4-independent recombinant envelope protein showed an increased binding of anti-envelope protein antibodies, suggesting either an enhanced binding to cell surfaces or more extensive conformational changes in CD4-independent compared to CD4-dependent envelope proteins. The reduced activation threshold of CD4-independent envelope proteins may thus enable them to utilize a membrane molecule for entry which is not as efficient as CD4 in triggering the conformational changes required for the membrane fusion process. CD4-independent HIV-2 variants may be conceptually similar to influenza virus variants capable of fusing at a higher than normal pH (R. S. Daniels, J. C. Downie, J. A. Hay, M. Knossow, J. J. Skehel, M. L. Wang, and D. C. Wiley, Cell 40:431-439, 1985).     

Unbiased research and the human spirit: the challenges of randomized controlled trials.

Research by Klein and associates provides useful information on the relation between episiotomy and outcomes such as perineal trauma, but the methodologic implications of their work are especially fascinating. Physicians who participated in their randomized controlled trial (RCT) were supposed to adhere to a policy of either liberal or restrictive use of episiotomy according to the study arm to which each patient was assigned. However, some used the procedure for approximately 90% of patients regardless of allocation. Klein and associates'' post-hoc study (see pages 769 to 779 of this issue) sheds light on the relation between physician attitudes and the practice of episiotomy. The author contends that the noncompliance encountered by Klein and associates reflects the fact that randomized trials are anathema to the human spirit. He offers suggestions for making RCTs more meaningful and stresses that, although RCTs are indispensible to the advancement of medical knowledge, they necessitate assiduous attention to matters of design and implementation.     

General relation between variance-time curve and power spectral density for point processes exhibiting 1/f β-fluctuations,with special reference to heart rate variability

Counting statistics in the form of the variance-time curve provides an alternative to spectral analysis for point processes exhibiting 1/f ^β-fluctuations, such as the heart beat. However, this is true only for β<1. Here, the case of general β is considered. To that end, the mathematical relation between the variance-time curve and power spectral density in the presence of 1/f ^β-noise is worked out in detail. A modified version of the variance-time curve is presented, which allows us to deal also with the case β?1. Some applications to the analysis of heart rate variability are given.     

Selective synthesis of depsipeptides by the immobilized multienzyme enniatin synthetase

Summary The multifunctional enzyme enniatin synthetase was immobilized by adsorption to propyl agarose. The immobilized multienzyme retained 45% of the activity of the free enzyme; an operational half-life of about 15 h was estimated. Selective synthesis of several different enniatin homologues was achieved with propyl agarose-bound enniatin synthetase. In addition to enniatin A, B, and C formation, a selective synthesis of non-naturally occurring depsipeptides, containing norvaline, norleucine, or -aminobutyric acid as sole amino acid moieties, was observed.     

Interaction of a glutathione S-conjugate with glutathione reductase. Kinetic and X-ray crystallographic studies

S-Conjugates of glutathione influence the glutathione/glutathione disulfide (GSH/GSSG) status of hepatocytes in at least two ways, namely by inhibition of GSSG transport into the bile [Akerboom et al. (1982) FEBS Lett. 140, 73-76] and by inhibition of the enzyme GSSG reductase (EC 1.6.4.2). The interaction of GSSG reductase with a well-studied conjugate, namely S-(2,4-dinitrophenyl)-glutathione and its electrophilic precursor 1-chloro-2,4-dinitrobenzene are described. For short exposures both compounds are reversible inhibitors of the enzyme, the Ki values being 30 microM and 22 microM respectively. After prolonged incubation, 1-chloro-2,4-dinitrobenzene blocks GSSG reductase irreversibly, which emphasizes the need for rapid conjugate formation in situ. As shown by X-ray crystallography the major binding site of S-(2,4-dinitrophenyl)-glutathione in GSSG reductase overlaps the binding site of the substrate, glutathione disulfide. However, the glutathione moiety of the conjugate does not bind in the same manner as either of the glutathiones in the disulfide.     

Isolation of an evolutionarily conserved epidermal growth factor receptor cDNA from human A431 carcinoma cells

Complementary DNA corresponding to total poly(A)+-RNA from the human A431 epidermoid carcinoma cell line was cloned in the phage expression vector lambda gt 11. An epidermal growth factor (EGF) receptor cDNA clone was obtained by screening of the expression library with a rabbit polyclonal antibody (IgG), raised to the purified A431 EGF receptor, in combination with [125I]protein A of S. aureus. The cloned cDNA was able to select, by hybridization, messenger RNA which was translated in Xenopus oocytes and yielded an immunoprecipitable EGF receptor protein of Mr = 160,000. The insert of this cDNA (phEGFR-1), is approximately 880 base pairs in length and encodes the carboxyterminal portion of the EGF receptor protein. Its sequence is evolutionarily conserved among vertebrates as shown by hybridization to unique chromosomal DNA sequences from human, baboon, dog, rat, mouse and frog.     

Mapping of the properdin-binding site in the third component of complement.

The properdin-binding site in the human third complement component (C3) was mapped by using isolated C3b, C3c, alpha- and beta-chains of C3 and C3 polypeptide fragments and an enzyme-linked-immunosorbent-assay procedure. The C3 chains and the polypeptide fragments were purified to homogeneity by preparative sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The alpha-chain polypeptides included a 68 kDa and a 43 kDa polypeptide, which were generated by cleavage of C3b with factors I and H, and a 40 kDa, 33 kDa (C3d) and 27 kDa polypeptide, which were generated by cleavage of C3b with porcine elastase. It was shown that properdin binds to C3b, C3c, alpha-chain, and to the 43 kDa (factor-I + H-derived), as well as to 40 kDa (elastase-derived) alpha-chain fragment, but not to the beta-chain 68 kDa, 33 kDa (C3d) and 27 kDa alpha-chain fragments. Thus the binding site for properdin resides on the 40-43 kDa C-terminal alpha-chain fragment of C3.     

Delayed tumor growth caused by a combined treatment with BCG and Pseudomonas aeruginosa

Summary Sera from mice treated i.v. with 1 mg BCG, followed 14 days later by 0.1 ml (10⁸ killed organisms) of Pseudomonas aeruginosa have shown the capacity to induce tumor necrosis when injected into mice bearing subcutaneous transplants either of a methyl-cholanthrene-induced sarcoma or of the P815 mastocytoma. Furthermore, immunotherapeutic trials were performed in mice bearing a subcutaneous transplanted sarcoma by combining BCG and low doses (0.01 to 0.05 ml) of Pseudomonas. Tumor necrosis was detectable 24 hours later only in the group treated by both BCG and Pseudomonas. In this group, we have also observed a significant decrease of tumor size in comparison with the groups of mice receiving BCG or Pseudomonas alone or no treatment.     

Refined mapping of the gene for glutathione reductase on human chromosome 8

Summary Activity of the enzyme glutathione reductase (EC 1.6.4.2) in erythrocytes and fibroblasts of a patient with karyotype 46, XY, del(8) (pterp212:) was found to be in the normal range. With results from other laboratories, this allowed a more precise mapping of the gene for this enzyme in the region 8p2100–8p212.