Regulation of apoptosis by BH3 domains in a cell-free system

Background: The Bcl-2 family of proteins plays a key role in the regulation of apoptosis. Some family members prevent apoptosis induced by a variety of stimuli, whereas others promote apoptosis. Competitive dimerisation between family members is thought to regulate their function. Homologous domains within individual proteins are necessary for interactions with other family members and for activity, although the specific mechanisms might differ between the pro-apoptotic and anti-apoptotic proteins.Results: Using a cell-free system based on extracts of Xenopus eggs, we have investigated the role of the Bcl-2 homology domain 3 (BH3) from different members of the Bcl-2 family. BH3 domains from the pro-apoptotic proteins Bax and Bak, but not the BH3 domain of the anti-apoptotic protein Bcl-2, induced apoptosis in this system, as determined by the rapid activation of specific apoptotic proteases (caspases) and by DNA fragmentation. The apoptosis-inducing activity of the BH3 domains requires both membrane and cytosolic fractions of cytoplasm, involves the release of cytochrome c from mitochondria and is antagonistic to Bcl-2 function. Short peptides, corresponding to the minimal sequence of BH3 domains required to bind anti-apoptotic Bcl-2 family proteins, also trigger apoptosis in this system.Conclusions: The BH3 domains of pro-apoptotic proteins are sufficient to trigger cytochrome c release, caspase activation and apoptosis. These results support a model in which pro-apoptotic proteins, such as Bax and Bak, bind to Bcl-2 via their BH3 domains, inactivating the normal ability of Bcl-2 to suppress apoptosis. The ability of synthetic peptides to reproduce the effect of pro-apoptotic BH3 domains suggests that such peptides may provide the basis for engineering reagents to control the initiation of apoptosis.     

Human alveolar macrophage fibronectin: synthesis, secretion, and ultrastructural localization during gelatin-coated latex particle binding

Human pulmonary alveolar macrophages synthesized and secreted several characteristic high molecular weight proteins for at least 7 d in vitro. Immunoprecipitates of medium and cell lysates from metabolically labeled cultures with specific anti-human plasma fibronectin IgG contained one major labeled polypeptide of molecular weight 440,000 (unreduced) or 220,000 (reduced). An identical polypeptide in conditioned medium from radiolabeled macrophages bound specifically to gelatin-Sepharose, demonstrating that alveolar macrophages synthesized and secreted a molecule immunologically and functionally similar to fibronectin. Fibronectin was the major newly synthesized and secreted polypeptide of freshly harvested alveolar macrophages. Pulse-chase experiments revealed that newly synthesized fibronectin was rapidly secreted into medium, approximately 50 percent appearing by 1 h and 80 percent by 8 h. Immunoperoxidase staining using antifibronectin F(ab’)(2)-peroxidase conjugates revealed the majority of immunoreactive fibronectin to be intracellular, localized to endoplasmic reticulum and Golgi apparatus. No extracellular matrix fibronectin was visualized, and cell surface staining was rarely seen, usually appearing only at sites where cells were closely apposed and not at sites of macrophage-substrate attachment. Similar immunostaining of fibroblast cultures revealed cell surface-associated fibrillar fibronectin. Ultrastructural localization of fibronectin during binding and phagocytosis of gelatin-coated and plain latex particles revealed fibronectin only on gelatin-latex beads and at their cell binding sites. Neigher plain latex beads nor their cell membrane binding sites stained for fibronectin. These results demonstrate that fibronectin is a major product of human alveolar macrophages, is rapidly secreted, and is localized at cell membrane binding sites for gelatin-coated particles. In view of the known binding properties of fibronectin, it may serve as an endogenous opsonic factor promoting the binding of staphylococcus, denatured collagen, fibrin, or other macromolecules to macrophages in the lower respiratory tract.     

Phylogenetic screening of the human genome: identification of differentially hybridizing repetitive sequence families

The phi-screen, a method of phylogenetic screening, can be employed to detect repetitive sequence families that differentially hybridize between closely related species. Such differences may involve sequence divergence or variations in copy number, including total presence versus absence of a family of repeated DNA. We present the results of a phi-screen comparing the human genome to that of the prosimian, Galago crassicaudatus. Three human repetitive families that are divergent or not present in galago have been detected. One of these families is described in detail; it is similar among the anthropoids but is present in a lower copy number and/or divergent form in prosimians. The family is clearly related to the transposon-like human element (THE) described by Paulson et al. (1985). THEs have long terminal repeats reminiscent of retroviruses but are unique in that they have no sequence similarity to known mammalian retroviruses. The sequence of a solo long terminal repeat, found unassociated with THE internal sequence, is presented. This family member, THE p2, is bordered by a 5-bp target-site repeat and is interrupted by the insertion of an Alu element. A solo THE element sequenced by Wiginton et al. (1986) contains an insertion of Alu at precisely the same position as does THE p2.      

DNA divergence in and around the alcohol dehydrogenase locus in five closely related species of Hawaiian Drosophila

The alcohol dehydrogenase (Adh) region from five planitibia subgroup species of Hawaiian picture-wing Drosophila has been cloned. A total of 15 kb of DNA in and around the Adh gene has been compared among the five species. Genetic distances were calculated to determine evolutionary relationships. These distances agree with previous distances determined by protein polymorphism and DNA hybridization techniques and can be interpreted in terms of specific island colonization and speciation (founder) events over the past 5 Myr. Examination of the restriction maps of the cloned Adh region from the five species shows many instances of small deletions, insertion of a transposable element in D. heteroneura, and the existence of a highly variable region on the 3' side of the Adh gene. Clustering relationships and rates of DNA change are calculated and compared with the relationship found for other species of Drosophila.      

Structure of the gene for human von Willebrand factor

von Willebrand factor is a large multimeric plasma protein composed of identical subunits which contain four types of repeated domains. von Willebrand factor is essential for normal hemostasis, and deficiency of von Willebrand factor is the most common inherited bleeding disorder of man. Four human genomic DNA cosmid libraries and one bacteriophage lambda library were screened with von Willebrand factor cDNA probes. Twenty positive overlapping clones were characterized that span the entire von Willebrand factor gene. A high-resolution restriction map was constructed for approximately 75% of the locus and a total of approximately 33.8 kilobases was sequenced on both strands including all intron-exon boundaries. The gene is approximately 178 kilobases in length and contains 52 exons. The exons vary from 40 to 1379 base pairs in length, and the introns vary from 97 base pairs to approximately 19.9 kilobases in length. The signal peptide and propeptide (von Willebrand antigen II) of von Willebrand factor are encoded by 17 exons in approximately 80 kilobases of DNA while the mature subunit of von Willebrand factor and 3' noncoding region are encoded by 35 exons in the remaining approximately 100 kilobases of the gene. A number of repetitive sequences were identified including 14 Alu repeats and a approximately 670-base pair TCTA simple repeat in intron 40 that is polymorphic. Regions of the gene that encode homologous domains have similar structures, supporting a model for their origin by gene segment duplication.