Cell engulfment, no sooner ced than done.

Three genes, ced-2, ced-5, and ced-10, are required for both cell corpse engulfment and distal tip cell migration in C. elegans. Recently, a fourth gene, ced-12, has been identified that is required for these two processes. ced-12 encodes a novel, conserved adaptor molecule involved in the activation of Rho/Rac/CDC42-like GTPases.

G-protein ligands inhibit in vitro reactions of vacuole inheritance.

During budding in Saccharomyces cerevisiae, maternal vacuole material is delivered into the growing daughter cell via tubular or vesicular structures. One of the late steps in vacuole inheritance is the fusion in the bud of vesicles derived from the maternal vacuole. This process has been reconstituted in vitro and requires isolated vacuoles, a physiological temperature, cytosolic factors, and ATP (Conradt, B., J. Shaw, T. Vida, S. Emr, and W. Wickner. 1992. J. Cell Biol. 119:1469-1479). We now report a simple and reliable assay to quantify vacuole-to-vacuole fusion in vitro. This assay is based on the maturation and activation of vacuole membrane-bound pro-alkaline phosphatase by vacuolar proteinase A after vacuole-to-vacuole fusion. In vitro fusion allowed maturation of 30 to 60% of pro-alkaline phosphatase. Vacuoles prepared from a mutant defective in vacuole inheritance in vivo (vac2-1) were inactive in this assay. Vacuole fusion in vitro required a vacuole membrane potential. Inhibition by nonhydrolyzable guanosine derivatives, mastoparans, and benzalkonium chloride suggest that GTP-hydrolyzing G proteins may play a key role in the in vitro fusion events.

Determination of four biochemically distinct, sequential stages during vacuole inheritance in vitro.

Vacuole inheritance in Saccharomyces cerevisiae can be reconstituted in vitro using isolated organelles, cytosol, and ATP. Using the requirements of the reaction and its susceptibility to inhibitors, we have divided the in vitro reaction into four biochemically distinct, sequential subreactions. Stage I requires exposure of vacuoles to solutions of moderate ionic strength. Stage II requires "stage I" vacuoles and cytosol. In stage III, stage II vacuoles react with ATP. Finally, during stage IV, stage III vacuoles at a certain, minimal concentration complete the fusion reaction without further requirement for any soluble components. Reagents that inhibit the overall vacuole inheritance reaction block distinct stages. Stage III of the reaction is sensitive to the proton ionophore CCCP, to inhibitors of the vacuolar ATPase such as bafilomycin A1, and to the ATP-hydrolyzing enzyme apyrase, suggesting that an electrochemical potential across the vacuolar membrane is required during this stage. Inhibition studies with the amphiphilic peptide mastoparan and GTP gamma S suggest that GTP-hydrolyzing proteins might also be involved during this stage. Microcystin-LR, a specific inhibitor of protein phosphatases of type 1 and 2A, inhibits stage IV of the inheritance reaction, indicating that a protein dephosphorylation event is necessary for fusion. The definition of these four stages may allow the development of specific assays for the factors which catalyze each of the consecutive steps of the in vitro reaction.

In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae.

Vacuole inheritance is temporally coordinated with the cell cycle and is restricted spatially to an axis between the maternal vacuole and the bud. The new bud vacuole is founded by a stream of vacuole-derived membranous vesicles and tubules which are transported from the mother cell into the bud to form the daughter organelle. We now report in vitro formation of vacuole-derived tubules and vesicles. In semi-intact cells, formation of tubulovesicular structures requires ATP and the proteins encoded by VAC1 and VAC2, two genes which are required for vacuole inheritance in vivo. Isolation of vacuoles from cell lysates before in vitro incubation reveals that formation of tubulovesicular structures requires cytosol as well as ATP. After forming tubulovesicular structures, isolated vacuoles subsequently increase in size. Biochemical assays reveal that this increase results from vacuole to vacuole fusion, leading to mixing of organellar contents. Intervacuolar fusion is sensitive to the phosphatase inhibitors microcystin-LR and okadaic acid, suggesting that protein phosphorylation/dephosphorylation reactions play a role in this event.

Translocation of C. elegans CED-4 to nuclear membranes during programmed cell death.

The Caenorhabditis elegans Bcl-2-like protein CED-9 prevents programmed cell death by antagonizing the Apaf-1-like cell-death activator CED-4. Endogenous CED-9 and CED-4 proteins localized to mitochondria in wild-type embryos, in which most cells survive. By contrast, in embryos in which cells had been induced to die, CED-4 assumed a perinuclear localization. CED-4 translocation induced by the cell-death activator EGL-1 was blocked by a gain-of-function mutation in ced-9 but was not dependent on ced-3 function, suggesting that CED-4 translocation precedes caspase activation and the execution phase of programmed cell death. Thus, a change in the subcellular localization of CED-4 may drive programmed cell death.

C. elegans ced-13 can promote apoptosis and is induced in response to DNA damage.

The p53 tumor suppressor promotes apoptosis in response to DNA damage. Here we describe the Caenorhabditis elegans gene ced-13, which encodes a conserved BH3-only protein. We show that ced-13 mRNA accumulates following DNA damage, and that this accumulation is dependent on an intact C. elegans cep-1/p53 gene. We demonstrate that CED-13 protein physically interacts with the antiapoptotic Bcl-2-related protein CED-9. Furthermore, overexpression of ced-13 in somatic cells leads to the death of cells that normally survive, and this death requires the core apoptotic pathway of C. elegans. Recent studies have implicated two BH3-only proteins, Noxa and PUMA, in p53-induced apoptosis in mammals. Our studies suggest that in addition to the BH3-only protein EGL-1, CED-13 might also promote apoptosis in the C. elegans germ line in response to p53 activation. We propose that an evolutionarily conserved pathway exists in which p53 promotes cell death by inducing expression of two BH3-only genes.

The efficiency of removal of lead and other elements from domestic drinking waters using a bench-top water filter system.

The efficiency of removal of lead (Pb) and other elements from natural drinking waters using a bench-top water filter system was evaluated in three recently-built houses in Sydney, Australia, and two from rural centres. In addition, one filter system was tested for copper (Cu), Pb and cadmium (Cd) under rigorously-controlled laboratory conditions using Sydney water. For two Sydney houses, the efficiency was evaluated using special filter cartridges concomitant with the ordinary filters. Waters after passing through the filter, was sampled when the filter had been exposed to '0', 50, 100, 150, 200, and 250 l respectively and were analysed for lead isotopes and lead concentrations by high precision isotope dilution mass spectrometry. Samples that passed through the filter after '0', 50, 150 and 250 l collections were analysed for four anions and 39 cations by various methods. Sydney water was fairly uniform in its anion and cation composition, whereas water from the two rural areas contained higher concentrations of Ca, Mg and HCO3. Effects of the filter on the water compositions can be summarised into three groups: (1) elements removed during filtration--Ca, Mg, Sr, Ba, Cu, Pb, Zn and Ni. With 'age' of the filter, the efficiency of removal for Pb was maintained in the two houses with Pb concentrations greater than 10 micrograms/l in the first flush water and was maintained for Cu through all concentrations. Ca, Mg, Sr and Ba were largely removed in the first 50 1 of usage. (2) Elements added during filtration--K, Rb, Ag and P. Except for Ag, which was present throughout the testing period, these elements were added only in the first filtration. (3) Elements unaffected by filtration--Al, Si, Na, Fe, Cl and F. Efficiency of Pb removal from tap water by this system depends generally on the initial Pb content in the water. However, it also seems to depend, to some extent, on Pb speciation and water composition, as found in earlier studies of natural waters. The control in filtering efficiency as a function of Pb speciation and chemical composition becomes more important at low initial Pb concentrations in the water. However, the Pb concentrations at which speciation and water composition appear to play a role in filter efficiency are so low that these factors, though scientifically interesting, have no relevance from a practical point of view, especially the impact on human health. Acidification of six sets of duplicate water samples, one sample immediately after collection and the other acidified after more than 2 weeks, showed negligible changes in Pb or Cu concentrations between duplicates.

Transcriptional upregulation of both egl-1 BH3-only and ced-3 caspase is required for the death of the male-specific CEM neurons.

Most of the 131 cells that die during the development of a Caenorhabditis elegans hermaphrodite do so approximately 30 min after being generated. Furthermore, in these cells, the pro-caspase proCED-3 is inherited from progenitors and the transcriptional upregulation of the BH3-only gene egl-1 is thought to be sufficient for apoptosis induction. In contrast, the four CEM neurons, which die in hermaphrodites, but not males, die approximately 150 min after being generated. We found that in the CEMs, the transcriptional activation of both the egl-1 and ced-3 gene is necessary for apoptosis induction. In addition, we show that the Bar homeodomain transcription factor CEH-30 represses egl-1 and ced-3 transcription in the CEMs, thereby permitting their survival. Furthermore, we identified three genes, unc-86, lrs-1, and unc-132, which encode a POU homeodomain transcription factor, a leucyl-tRNA synthetase, and a novel protein with limited sequence similarity to the mammalian proto-oncoprotein and kinase PIM-1, respectively, that promote the expression of the ceh-30 gene in the CEMs. On the basis of these results, we propose that egl-1 and ced-3 transcription are coregulated in the CEMs to compensate for limiting proCED-3 levels, which most probably are a result of proCED-3 turn over. Similar coregulatory mechanisms for BH3-only proteins and pro-caspases may function in higher organisms to allow efficient apoptosis induction. Finally, we present evidence that the timing of the death of the CEMs is controlled by TRA-1 Gli, the terminal global regulator of somatic sexual fate in C. elegans.

egl-1: a key activator of apoptotic cell death in C. elegans.

Since the discovery of mammalian BIK and BAD in 1995, BH3-only proteins have emerged as key activators of apoptotic cell death in animals as diverse as the nematode, Caenorhabditis elegans, and humans. BH3-only proteins have also emerged as integrators of cell-death signals that determine the life-versus-death decision and that transduce this decision to the central apoptotic machinery through their physical interaction with 'core' BCL-2 family members, such as BCL-2 or BCL-XL. Currently, eight BH3-only proteins have been identified and characterized in mammals, and there is evidence of functional overlap between them. In contrast, only two BH3-only proteins have so far been identified and characterized in C. elegans, EGL-1 and CED-13, and there seems to be only limited functional overlap between them. Combined with the powerful genetic tools available for the analysis of apoptosis in C. elegans, and the ability to study apoptosis at single-cell resolution in this organism, the absence of extensive functional redundancy makes C. elegans an ideal model for studies on BH3-only proteins. In this study, we will review our current understanding of the role and regulation of EGL-1. We will also briefly summarize studies on CED-13, which was identified more recently.

A truncated form of the Pho80 cyclin of Saccharomyces cerevisiae induces expression of a small cytosolic factor which inhibits vacuole inheritance.

Vacuoles project streams of vesicles and membranous tubules into the yeast bud where they fuse, founding the daughter cell organelle, vac5-1, which encodes a truncated form of the Pho80 cyclin, inhibits normal vacuole inheritance. An in vitro inheritance assay which measures the fusion of vacuoles serves as a model for several steps of this process. We find that cytosol isolated from the vac5-1 mutant is unable to promote the fusion of wild-type vacuoles in the in vitro assay. Wild-type vacuoles are irreversibly inactivated in a time- and temperature-dependent manner if preincubated with vac5-1 cytosol and ATP, suggesting the presence of a soluble inhibitory factor. When mixed with wild-type cytosol, vac5-1 cytosol inhibits the activity of wild-type cytosol. vac5-1 cytosol treated with trypsin or papain is still able to inhibit the activity of Aid-type cytosol. Partial fractionation of vac5-1 cytosol reveals that the protein traction (G25 void volume) can promote fusion if wild-type small molecules are included in the fusion reaction. In contrast, the vac5-l small-molecule fraction retains the full ability to inhibit fusion. Thus, the vac5-1 allele of PHO80 induces the synthesis of a small molecule that is an inhibitor of vacuole inheritance.

The TRA-1A sex determination protein of C. elegans regulates sexually dimorphic cell deaths by repressing the egl-1 cell death activator gene.

The hermaphrodite-specific neurons (HSNs) of the nematode Caenorhabditis elegans are generated embryonically in both hermaphrodites and males but undergo programmed cell death in males. The gene egl-1 encodes a BH3-containing cell death activator that is required for programmed cell death in C. elegans. Gain-of-function (gf) mutations in egl-1 cause the inappropriate programmed cell death of the HSNs in hermaphrodites. These mutations lie 5.6 kb downstream of the egl-1 transcription unit and disrupt the binding of the TRA-1A zinc finger protein, the terminal global regulator of somatic sexual fate. This disruption results in the activation of the egl-1 gene in the HSNs not only in males but also in hermaphrodites. Our findings suggest that in hermaphrodites TRA-1A represses egl-1 transcription in the HSNs to prevent these neurons from undergoing programmed cell death.

The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl-2-like protein CED-9.

Gain-of-function mutations in the Caenorhabditis elegans gene egl-1 cause the HSN neurons to undergo programmed cell death. By contrast, a loss-of-function egl-1 mutation prevents most if not all somatic programmed cell deaths. The egl-1 gene negatively regulates the ced-9 gene, which protects against cell death and is a member of the bcl-2 family. The EGL-1 protein contains a nine amino acid region similar to the Bcl-2 homology region 3 (BH3) domain but does not contain a BH1, BH2, or BH4 domain, suggesting that EGL-1 may be a member of a family of cell death activators that includes the mammalian proteins Bik, Bid, Harakiri, and Bad. The EGL-1 and CED-9 proteins interact physically. We propose that EGL-1 activates programmed cell death by binding to and directly inhibiting the activity of CED-9, perhaps by releasing the cell death activator CED-4 from a CED-9/CED-4-containing protein complex.

Immunohistochemical detection of progesterone receptor in formalin-fixed and paraffin-embedded breast cancer tissue using a monoclonal antibody.

The potential for immunohistochemical detection of progesterone receptors (PRs) in routinely formalin-fixed and paraffin-embedded cancer tissues by use of the monoclonal antibody Mi 60-10 (mPR1, Dianova GmbH, Hamburg) was evaluated. The PR content of breast cancer tissue was investigated in 170 cases. A positive reaction to Mi 60-10 was found exclusively in the nuclei of benign or malignant epithelial cells. The distribution of PRs was heterogeneous. Immunohistochemical reaction was scored by multiplying the percentage of positive tumour cells by their prevalent degree of staining (Immunoreactive Score or IRS). The IRS values of formalin-fixed tissues (n = 170) were compared with those in snap frozen tissues (n = 82), with the PR content assayed by a DCC (dextran-coated charcoal) method (n = 170), with histopathological grading according to Bloom and Richardson and with the menopausal status of the patient. There was an acceptable ranked correlation (r = 0.74) between IRS in formalin-fixed and paraffin-embedded parts and snap frozen parts of the same carcinoma. A good correlation (r = 0.72) was also found, when the semiquantitative results of immunohistochemical PR detection in formalin-fixed and paraffin-embedded tissues were compared to PR concentrations measured by a DCC method in tumor cytosols. There was an 80% concordance between the two methods for qualitative discrimination of PR-negative and PR-positive carcinomas. IRS correlated significantly with the degree of histological differentiation of the tumors (P less than 0.001) but not with the menopausal status of the women (P greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)