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L M Schwartz

Publications and source records attributed to L M Schwartz.

At least 19 recordsLinked to original sources
PubMed1995

Programmed cell death in the Drosophila central nervous system midline.

BACKGROUND: During the development of the central nervous system, large numbers of cells die by programmed cell death. This process requires the activity of specific gene products and subserves functions that include regulating the sizes of interacting cell populations and removing cells that provide transient functions. Resolution of programmed cell death often involves the elimination of dying cell corpses by phagocytic macrophages. In Drosophila, the reaper gene plays a crucial role in mediating programmed cell death; chromosomal deficiencies which remove reaper result in an absence of programmed cell death. We have used a reaper-deficiency mutant strain Df(3R)H99 (or H99), in conjunction with strains containing cell-type-specific markers, to examine the role of programmed cell death in differentiation of the embryonic central nervous system midline. RESULTS: Midline cell death was identified both by the presence of excess midline cells in H99 mutants and by the engulfment of dying midline cells by macrophages in wild-type embryos. These developmental deaths are lineage-specific: prominent midline glial death was observed, while little if any death was detected among the ventral unpaired median neurons. Examination of H99 mutants indicates that cell death is not required for the formation of macrophage precursors, or for their subsequent migration throughout the embryo; however, in the absence of dying cells, macrophage precursors do not exhibit morphological differentiation or phagocytosis. In both wild-type and H99 mutant embryos, a subset of macrophages migrate along the ventral midline. This midline migration is not observed in single-minded mutants, in which ventral midline cells fail to develop. CONCLUSIONS: Programmed cell death plays a crucial role in the development of the central nervous system midline, and dying midline cells are rapidly eliminated by phagocytic macrophages. It seems that the generation of engulfment signals in cells undergoing programmed cell death is downstream of reaper gene function, and that central nervous system midline and/or ventral epidermal cells provide directional cues for migrating macrophages.

Animals↗
PubMed1995

Coordinated induction of the ubiquitin conjugation pathway accompanies the developmentally programmed death of insect skeletal muscle.

The developmentally programmed cell death of abdominal intersegmental muscles in the tobacco hawk-moth Manduca sexta is coincident with a 10-fold induction of the polyubiquitin gene as a hormonally regulated event (Schwartz, L. M., Myer, A., Kosz, L., Engelstein, M., and Maier, C. (1990) Neuron 5, 411-419). Solid phase immunochemical assays measuring intersegmental muscle pools of free and conjugated ubiquitin reveal that the induction of polyubiquitin mRNA is accompanied by a proportional increase in total ubiquitin polypeptide. Ubiquitin conjugate pools increase 10-fold at eclosion, during which loss of muscle protein mass is maximum. A smaller but measurable increase in ubiquitin conjugates is observed earlier in pupal development coincident with a modest enhanced degradation of myofibrillar proteins. Accumulation of ubiquitin conjugates is accompanied by induction in the pathway for polypeptide ligation, including the activating enzyme (E1), several carrier protein (E2) isoforms, and ubiquitin:protein isopeptide ligase (E3). Both accumulation of ubiquitin polypeptide and the enzymes of the conjugation pathway are subject to regulation by declining titers of the insect molting hormone 20-hydroxyecdysone, which signals onset of programmed cell death in the intersegmental muscles. Thus, programmed cell death within the intersegmental muscles is accomplished in part by stimulation of the ubiquitin-mediated degradative pathway through a coordinated induction of ubiquitin and the enzymes responsible for its conjugation to yield proteolytic intermediates. This suggests enzymes required for ubiquitin conjugation may represent additional genes recruited for developmentally programmed death.

Animals↗
PubMed1995

The multicatalytic proteinase (proteasome) of the hawkmoth, Manduca sexta: catalytic properties and immunological comparison with the lobster enzyme complex.

The proteasome plays a central role in ubiquitin-dependent and -independent proteolysis in eukaryotic cells. The hawkmoth proteasome was purified from larval body wall and characterized with respect to substrate specificity, sensitivity to protease inhibitors, and cross-reactivity with monoclonal antibodies (mAbs) raised against human placenta proteasome. Leupeptin selectively inhibited the trypsin-like activity (T-L) and N-ethylmaleimide inhibited both T-L and chymotrypsin-like activities, whereas 0.02% sodium dodecyl sulfate stimulated the peptidylglutamyl peptide hydrolase, branched-chain amino acid preferring, and caseinolytic activities 20-, 18-, and 3.8-fold, respectively. All four peptidase activities were inhibited by 3,4-dichloroisocoumarin. One-dimensional immunoblot analysis showed that the level and subunit composition of the proteasome varied between tissues. The relative levels of proteasome were high in intersegmental muscle and ovary, lower in Malpighian tubule, male accessory gland, and ventral nerve cord, and lowest in flight muscle and fat body. The tissues differed in the relative amount of a 41-kDa doublet; a 22-kDa subunit was present only in the male accessory gland. Two-dimensional polyacrylamide gel electrophoresis showed that the hawkmoth proteasome contained at least 26 subunits, compared with 28 subunits in lobster. Immunological analysis using four subunit-specific mAbs identified the putative homologs of the human zeta, C2, C3, and C8 alpha-type subunits in the hawkmoth and lobster enzymes. Two of the four mAbs reacted with three or more of the hawkmoth subunits and three of the mAbs reacted with two or more of the lobster subunits. In addition, two other mAbs that recognize epitopes shared by a number of alpha-type subunits indicated that at least 15 (lobster) or 16 (hawkmoth) subunits were alpha-type. These results suggest that much of the subunit complexity of the arthropod proteasomes is a consequence of extensive post-translational modifications.

Amino Acid Sequence↗
PubMed1995

Cell death suffers a TKO.

The cytokine interferon-gamma (IFN-gamma), initiates both cell cycle arrest and cell death in certain cell lines. Through a novel strategy of cell transfection with episomal vectors expressing antisense cDNAs, Deiss et al. have demonstrated that it is possible to isolate genes that are required for the initiation of cell death by the cytokine IFN-gamma. This approach, referred to as TKO, for Technical Knock Out, has identified several genes whose activity appears to be essential for the induction of apoptosis by IFN-gamma in HeLa cells. Interestingly, these genes appear to mediate IFN-gamma-induced apoptosis in HeLa cells, but their inhibition by antisense does not ameliorate the antiproliferative effects of IFN-gamma in these cells. The clever strategy employed by these authors holds promise for others who wish to isolate genes required for other differentiative processes in cultured cell lines.

Antigens, Viral↗
PubMed1995

Ubiquitin in homeostasis, development and disease.

Ubiquitin is the most phylogenetically conserved protein known. This 8,500 Da polypeptide can be covalently attached to cellular proteins as a posttranslational modification. In most cases, the addition of multiple ubiquitin adducts to a protein targets it for rapid degradation by a multisubunit protease known as the 26S proteasome. While the ubiquitin/26S proteasome pathway is responsible for the degradation of the bulk of cellular proteins during homeostasis, it may also be responsible for the rapid loss of protein during the programmed death of certain cells, such as skeletal muscle during insect metamorphosis. In addition, alterations in the expression and regulation of ubiquitin may play significant roles in pathological disorders. For example, dramatic increases in ubiquitin and ubiquitin-protein conjugates are observed in a wide variety of neurodegenerative disorders, including Alzheimer's disease. Patients suffering from the autoimmune disease systemic lupus erythematosus generate antibodies reacting with ubiquitin and ubiquitinated histones. At present, it is not known whether these changes in ubiquitin expression and regulation initiate pathological changes in these diseases or if they are altered as a consequence of these disorders.

Animals↗
PubMed1995

Apolipophorin III is dramatically up-regulated during the programmed death of insect skeletal muscle and neurons.

The intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta, participate in the emergence behavior of the adult moth and then die during the subsequent 30 hours. In addition, several populations of interneurons and uniquely identified motor neurons also die after adult emergence. The trigger for all of these deaths is a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone. The ability of the muscles and neurons to die requires de novo gene expression. A differential hybridization screen of a "condemned" ISM cDNA library permitted the isolation of clones encoding four new up-regulated mRNAs. On sequencing, one of these recombinants was found to encode apolipophorin III (apoLp-III), a component of lipophorin, the major hemolymph lipoprotein of insects, previously shown to be synthesized in fat body. Although apoLp-III mRNA and protein were expressed at all stages of ISM development, levels of both molecules were dramatically elevated with the commitment of the cells to die. When ISM cell death was delayed by injection of 20-hydroxyecdysone, expression of apoLp-III at both the RNA and protein levels was markedly reduced at the normal time of cell death. Immunocytochemistry demonstrated that apoLp-III protein was abundantly expressed in the cytoplasm of dying muscles, interneurons, and identified motor neurons at the time of cell death. Apolipoproteins I and II, required components of lipophorin, were not expressed at detectable levels in the muscles or neurons. Furthermore, Western blots of native gels suggest that apoLp-III was not associated with any other proteins. These data suggest that apoLp-III has activities independent of lipid transport that may play a role in programmed cell death. ApoLp-III joins apolipoproteins E and J (clusterin, sulfated glycoprotein-2) as a group of proteins that function in both lipid transfer and cell death.

Animals↗
PubMed1995

Changes in the structure and function of the multicatalytic proteinase (proteasome) during programmed cell death in the intersegmental muscles of the hawkmoth, Manduca sexta.

The intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta are a well-characterized model system for examining the biochemical changes that accompany programmed cell death during development. These giant muscles die during a 30-hr period in response to a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone. When the ISMs become committed to die, there are dramatic increases in both ubiquitin expression and ubiquitin-dependent proteolysis. Since the multicatalytic proteinase (MCP) is responsible for ATP/ubiquitin-dependent proteolysis in cells, we examined its composition and properties. The purified enzyme from whole larval integumentary tissues resembles MCPs isolated from other species with respect to subunit composition and general catalytic properties. However, when MCP was isolated from condemned ISMs, we observed an approximately ninefold increase in proteinase activity compared to MCP from precommitment muscles. This increase in proteolytic activity was correlated with an approximately eightfold increase in the absolute amounts of MCP protein as determined by Western blotting and densitometry. When purified MCP from condemned muscles was examined by two-dimensional polyacrylamide gel electrophoresis, four new subunits that were not detected in the precommitment muscles were present. Correlated with the addition of these new subunits was a dramatic increase in the levels of immunodetectable MCP throughout the cytoplasm and within the nuclei of dying muscles. These changes in MCP were regulated by the same hormonal signals that mediate cell death. These data are consistent with the hypothesis that when the ISMs become committed to die, more MCP accumulates in cells and new subunits are synthesized that change both the enzymatic properties and the conformation of MCP, which in turn participates in the dramatic proteolysis that accompanies cell death.

Amino Acid Sequence↗
PubMed1995

Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in vitro.

Members of the CED-3/interleukin-1 beta-converting enzyme (ICE) protease family have been implicated in cell death in both invertebrates and vertebrates. In this report, we show that peptide inhibitors of ICE arrest the programmed cell death of motoneurons in vitro as a result of trophic factor deprivation and in vivo during the period of naturally occurring cell death. In addition, interdigital cells that die during development are also rescued in animals treated with ICE inhibitors. Taken together, these results provide the first evidence that ICE or an ICE-like protease plays a regulatory role not only in vertebrate motoneuron death but also in the developmentally regulated deaths of other cells in vivo.

Amino Acid Sequence↗
PubMed1995

Transient transfection assays to examine the requirement of putative cell death genes.

In conclusion, this chapter provides a convenient and efficient method for the detection and analysis of transiently transfected cells. Such strategies allow a fast and simple analysis of the requirement for particular genes that have been identified as being induced during apoptosis. Our experience has been that, when screening for "cell death genes," it is easy to isolate genes induced during apoptosis but far more difficult to determine the requirement for any given gene. These protocols have rendered such determinations much simpler to perform.

Antigens, CD↗
PubMed1995

Cell death.

Explore the source record for details and available documents.

Animals↗
PubMed1994

Localization of immunoreactive ubiquitin in the nervous system of the Manduca sexta moth.

Selective neuronal death is a normal component of metamorphosis in the moth, Manduca sexta. In particular, the three unfused abdominal ganglia of the ventral nerve cord serve as a useful experimental preparation in which to study the regulation of the molecular mechanisms that mediate programmed cell death. Ubiquitin, a highly conserved 76-amino acid protein found in all eukaryotic cells, has previously been shown to be present in increased amounts in some tissues undergoing programmed cell death (e.g., larval intersegmental muscles in Manduca sexta moths, dying cells in developing tunicates), but not in others (T-cells, Drosophila ommatidial cells, cultured sympathetic neurons deprived of nerve growth factor). It has been hypothesized that the need for ubiquitin-dependent proteolysis is increased in dying cells, and that the accumulation of ubiquitin might serve as an early marker for cells committed to die. Immunohistochemical localization of ubiquitin at the light microscopic level in the abdominal ganglia of Manduca sexta suggests that this protein plays a number of important roles in neuronal physiology and may be associated with the death of some neurons in this tissue. The most intense staining of neuronal cytoplasm, however, was found not in dying neurons, but instead in sets of persisting neurons that may serve a primarily neurosecretory or neuromodulatory function. The staining obtained in these cells with antibodies directed against ubiquitin was developmentally regulated.

Abdomen↗
PubMed1994

Apoptotic signals delivered through the T-cell receptor of a T-cell hybrid require the immediate-early gene nur77.

Engagement of the T-cell antigen receptor (TCR) on immature thymic T cells induces death by apoptosis. Although several lines of evidence indicate that apoptosis requires de novo gene expression, little is known about the molecular pathways that mediate this response. Here we show that nur77 (refs 4-7), a zinc-finger transcription factor, is expressed in response to TCR engagement in immature T cells and T-cell hybrids. Antisense inhibition of nur77 expression prevents apoptosis in TCR-stimulated cells. nur77 is also expressed in response to mitogens, but in this case transcription is regulated by 5' upstream elements that are distinct from those used for induction of apoptosis. In addition, polyadenylation is only observed on nur77 transcripts found in condemned cells. These data support a role for nur77 in cell death that may be distinct from that of activation.

Animals↗
PubMed1994

Motoneurons deprived of trophic support in vitro require new gene expression to undergo programmed cell death.

During normal development, large numbers of neurons die by programmed cell death. This phenomena has been extensively studied in the lateral motor column of chick embryos, where approximately 50% of the motoneurons that are initially produced, subsequently die due in part to competition for a limited supply of target-derived trophic support. Inhibitors of RNA and protein synthesis block this cell loss in vivo, indicating a requirement for new gene expression (Oppenheim et al., 1990). Prior to their commitment to death, motoneurons can be isolated as a relatively pure population from chick spinal cord for in vitro study. Cells plated with muscle extract, a potent source of target-derived trophic support, survive, and have large, phase-bright cell bodies and extensive neurite outgrowth. In contrast, motoneurons cultured in the absence of muscle extract die within 48 h. This death can be blocked by the RNA synthesis inhibitor actinomycin D, at the time when the cells become committed to die, suggesting that new gene expression is required for cell death. DNA fragmentation and nuclear condensation indicate that some of these cells die by apoptosis. Therefore, it appears that many aspects of motoneuron development observed in vivo can be reconstituted in vitro. These cultures can be used as a model system for studying neuronal death and may contribute to an understanding of the molecular mechanisms that mediate programmed cell death during neuronal development.

Animals↗
PubMed1994

Probing the structure of porous media using NMR spin echoes.

The NMR pulsed field gradient spin-echo (PFGSE) amplitude M(k,t) is a direct measure of the diffusion propagator of the molecules of a fluid diffusing in the pores of a porous medium. For small values of k = gamma g delta, where g is the gradient strength, delta is the duration of the gradient pulse, and gamma the gyromagnetic ratio, the PFGSE amplitude gives the diffusion coefficient D(t). The exact short-time diffusion coefficient, D(t)/D0 = 1-(4 square root of D0tS)/(9 Vp square root of pi)-D0tHS/(12Vp) + rho tS/6Vp, provides an important method for determining the surface to pore-volume ratio S/Vp. Here the mean surface curvature H = <1/R1 + 1/R2>. Combining early D(t) with the magnetization decay one obtains the surface relaxivity rho. The long-time effective diffusion constant derived from PFGSE gives information on the tortuosity of the connected space. The diffusion coefficient measured by PFGSE equals that derived from electrical conductivity only when rho = 0. Exact solutions with partially absorbing boundary conditions for a periodic structure are used to illustrate the influence of rho on the diffusion coefficient. M(k,t) can be well represented by a convolution of the structure factor of the connected pore space with an appropriate Gaussian propagator. This ansatz provides a model-independent way of obtaining the structure factor.

Diffusion↗
PubMed1994

Simulations of pulsed field gradient spin-echo measurements in porous media.

Under ideal conditions, in an isotropic homogeneous system, pulsed field gradient spin-echo (PFGSE) experiments measure the probability P(R,t), that a diffusing water molecule will travel a root mean square distance, R, in a time interval of length t. We will show that random walk simulations on model porous media provide valuable insight to the sensitivity of this measurement to (1) complex pore geometries and (2) enhanced relaxation at the pore-grain interface.

Computer Simulation↗