Constitutive Bcl-2 expression throughout the hematopoietic compartment affects multiple lineages and enhances progenitor cell survival

Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous overexpression within this system, we targeted expression of the human bcl-2 gene in mice by using the promoter of the vav gene, which is active throughout this compartment but rarely outside it. The vav-bcl-2 transgene was expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice displayed increases in myeloid cells as well as a marked elevation in B and T lymphocytes. The spleen was enlarged and the lymphoid follicles expanded. Although total thymic cellularity was normal, T cell development was altered: cells at the very immature and most mature stages were increased, whereas those at the intermediate stage were decreased. Unexpectedly, blood platelets were reduced by half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells in vitro remained cytokine dependent, and the frequency of most progenitor and preprogenitor cells was normal. Macrophage progenitors were less frequent and yielded smaller colonies, however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhanced clonogenic survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the role of apoptosis in the development and homeostasis of this compartment.

Folding of Active β-Lactamase in the Yeast Cytoplasm before Translocation into the Endoplasmic Reticulum

Polypeptides targeted to the yeast endoplasmic reticulum (ER) posttranslationally are thought to be kept in the cytoplasm in an unfolded state by Hsp70 chaperones before translocation. We show here that Escherichia coli β-lactamase associated with Hsp70, but adopted a native-like conformation before translocation in living Saccharomyces cerevisiae cells. β-Lactamase is a globular trypsin-resistant molecule in authentic form. For these studies, it was linked to the C terminus of a yeast polypeptide Hsp150Δ, which conferred posttranslational translocation and provided sites for O-glycosylation. We devised conditions to retard translocation of Hsp150Δ-β-lactamase. This enabled us to show by protease protection assays that an unglycosylated precursor was associated with the cytoplasmic surface of isolated microsomes, whereas a glycosylated form resided inside the vesicles. Both proteins were trypsin resistant and had similar β-lactamase activity and Km values for nitrocefin. The enzymatically active cytoplasmic intermediate could be chased into the ER, followed by secretion of the activity to the medium. Productive folding in the cytoplasm occurred in the absence of disulfide formation, whereas in the ER lumen, proper folding required oxidation of the sulfhydryls. This suggests that the polypeptide was refolded in the ER and consequently, at least partially unfolded for translocation.

Induction of Exocytosis from Permeabilized Mast Cells by the Guanosine Triphosphatases Rac and Cdc42

We applied recombinant forms of the Rho-related small guanosine triphosphatases (GTPases) Rac2 and Cdc42/G25K to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca2+ and guanosine triphosphate (GTP)γS over about 20–30 min. This loss of sensitivity is likely to be due to loss of key regulatory proteins that are normally tethered at intracellular locations. Exogenous proteins that retard this loss of sensitivity to stimulation may be similar, if not identical, to those secretory regulators that are lost. Recombinant Rac and Cdc42/G25K, preactivated by binding GTPγS, retard the loss of sensitivity (run-down) and, more importantly, enable secretion to be stimulated by Ca2+ alone. Investigation of the concentration dependence of each of these two GTPases applied individually to the permeabilized cells, and of Cdc42/G25K applied in the presence of an optimal concentration of Rac2, has provided evidence for a shared effector pathway and also a second effector pathway activated by Cdc42/G25K alone. Dominant negative mutant (N17) forms of Rac2 and Cdc42/G25K inhibit secretion induced by Ca2+ and GTPγS. Our data suggest that Rac2 and Cdc42 should be considered as candidates for GE, GTPases that mediate exocytosis in cells of hematopoeitic origin.

HDA1 and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription

Increased histone acetylation has been correlated with increased transcription, and regions of heterochromatin are generally hypoacetylated. In investigating the cause-and-effect relationship between histone acetylation and gene activity, we have characterized two yeast histone deacetylase complexes. Histone deacetylase-A (HDA) is an ≈350-kDa complex that is highly sensitive to the deacetylase inhibitor trichostatin A. Histone deacetylase-B (HDB) is an ≈600-kDa complex that is much less sensitive to trichostatin A. The HDA1 protein (a subunit of the HDA activity) shares sequence similarity to RPD3, a factor required for optimal transcription of certain yeast genes. RPD3 is associated with the HDB activity. HDA1 also shares similarity to three new open reading frames in yeast, designated HOS1, HOS2, and HOS3. We find that both hda1 and rpd3 deletions increase acetylation levels in vivo at all sites examined in both core histones H3 and H4, with rpd3 deletions having a greater impact on histone H4 lysine positions 5 and 12. Surprisingly, both hda1 and rpd3 deletions increase repression at telomeric loci, which resemble heterochromatin with rpd3 having a greater effect. In addition, rpd3 deletions retard full induction of the PHO5 promoter fused to the reporter lacZ. These data demonstrate that histone acetylation state has a role in regulating both heterochromatic silencing and regulated gene expression.

Electrostatic surface plasmon resonance: Direct electric field-induced hybridization and denaturation in monolayer nucleic acid films and label-free discrimination of base mismatches

We demonstrate that in situ optical surface plasmon resonance spectroscopy can be used to monitor hybridization kinetics for unlabeled DNA in tethered monolayer nucleic acid films on gold in the presence of an applied electrostatic field. The dc field can enhance or retard hybridization and can also denature surface-immobilized DNA duplexes. Discrimination between matched and mismatched hybrids is achieved by simple adjustment of the electrode potential. Although the electric field at the interface is extremely large, the tethered single-stranded DNA thiol probes remain bound and can be reused for subsequent hybridization reactions without loss of efficiency. Only capacitive charging currents are drawn; redox reactions are avoided by maintaining the gold electrode potential within the ideally polarizable region. Because of potential-induced changes in the shape of the surface plasmon resonance curve, we account for the full curve rather than simply the shift in the resonance minimum.

Systemin Potentiates the Oxidative Burst in Cultured Tomato Cells1

Plants that have been wounded by insects or other herbivores may be more susceptible to infection by adventitious microbes. Wound-induced signal molecules, which serve to induce responses in the plant that retard further feeding, might also act to prepare a plant for possible pathogen attack. We have examined the effect of a wound-generated systemic messenger (systemin) on a pathogen-stimulated defense-response marker, the oxidative burst. We observed that neither systemin nor its inactive analog (A-17) was able to directly induce H2O2 biosynthesis in suspension-cultured tomato (Lycopersicon esculentum L.) cells, regardless of the duration of exposure of the cells to the two peptides. Similarly, neither systemin nor A-17 was capable of modifying an oligogalacturonide-elicited oxidative burst, as long as elicitor addition occurred within minutes of treatment with systemin or A-17. In contrast, preexposure of the cell cultures to systemin (but not to A-17) led to a time-dependent enhancement of the oligogalacturonide-elicited oxidative burst. By 12 h of exposure, the H2O2 biosynthetic capacity of systemin-treated cells exceeded that of the control cells by a factor of 16 ± 2. A similar up-regulation by systemin of a mechanically stimulated oxidative burst was also observed. Because the systemin-induced augmentation in oxidant synthesis is quantitatively prevented by coincubation with 2 μm cycloheximide, and because the oxidative burst of oligogalacturonic acid-elicited control cells (no systemin exposure) is unaffected by preincubation with cycloheximide, we conclude that systemin enhancement of the tomato-cell oxidative burst requires protein synthesis.

cAMP-response element modulator tau is a positive regulator of testis angiotensin converting enzyme transcription.

Testis angiotensin-converting enzyme (ACE) is a unique form of ACE, only produced by male germ cells, and results from a testis-specific promoter found within the ACE gene. We have investigated the role of cAMP-response element modulator (CREM)tau in testis ACE transcription. In gel shift experiments, testes nuclear proteins retard an oligonucleotide containing the cAMP-response element (CRE) found at position -55 in the testis ACE promoter. Anti-CREM antibody supershifts this complex. Competitive gel shift shows that recombinant CREM tau protein and testis nuclear proteins have a similar specificity of binding to the tests ACE CRE. Functional analysis using in vitro transcription and transfection studies also demonstrate that CREM tau protein is a transcriptional activator of the testis ACE promoter. Western blot analysis identifies CREM tau protein in the protein-DNA complex formed between nuclear proteins and the testis ACE CRE motif. This analysis also identified other CREM isoforms in the gel-shifted complex, which are thought to be CREM tau 1/2, CREM alpha/beta, and S-CREM. These data indicate that CREM tau isoforms play an important role as a positive regulator in the tissue-specific expression of testis ACE.

Protonatable residues at the cytoplasmic end of transmembrane helix-2 in the signal transducer HtrI control photochemistry and function of sensory rhodopsin I.

Neutral residue replacements were made of 21 acidic and basic residues within the N-terminal half of the Halobacterium salinarium signal transducer HtrI [the halobacterial transducer for sensory rhodopsin I (SRI)] by site-specific mutagenesis. The replacements are all within the region of HtrI that we previously concluded from deletion analysis to contain sites of interaction with the phototaxis receptor SRI. Immunoblotting shows plasmid expression of the htrI-sopI operon containing the mutations produces SRI and mutant HtrI in cells at near wild-type levels. Six of the HtrI mutations perturb photochemical kinetics of SRI and one reverses the phototaxis response. Substitution with neutral amino acids of Asp-86, Glu-87, and Glu-108 accelerate, and of Arg-70, Arg-84, and Arg-99 retard, the SRI photocycle. Opposite effects on photocycle rate cancel in double mutants containing one replaced acidic and one replaced basic residue. Laser flash spectroscopy shows the kinetic perturbations are due to alteration of the rate of reprotonation of the retinylidene Schiff base. All of these mutations permit normal attractant and repellent signaling. On the other hand, the substitution of Glu-56 with the isosteric glutamine converts the normally attractant effect of orange light to a repellent signal in vivo at neutral pH (inverted signaling). Low pH corrects the inversion due to Glu-56 -> Gln and the apparent pK of the inversion is increased when arginine is substituted at position 56. The results indicate that the cytoplasmic end of transmembrane helix-2 and the initial part of the cytoplasmic domain contain interaction sites with SRI. To explain these and previous results, we propose a model in which (i) the HtrI region identified here forms part of an electrostatic bonding network that extends through the SRI protein and includes its photoactive site; (ii) alteration of this network by photoisomerization-induced Schiff base deprotonation and reprotonation shifts HtrI between attractant and repellent conformations; and (iii) HtrI mutations and extracellular pH alter the equilibrium ratios of these conformations.

The timing of synaptic vesicle endocytosis.

Alternative models to describe the endocytosis phase of synaptic vesicle recycling are associated with time scales of vesicle recovery ranging from milliseconds to tens of seconds. There have been suggestions that one of the major models, envisioned as a slow process that occurs only after complete fusion of the vesicle membrane with the neurolemma, might be applicable only under conditions of heavy, nonphysiological stimulation. Using FM 1-43 and similar fluorescent probes to label recycling synaptic vesicles in rat hippocampal neurons, we have measured the kinetics of endocytosis with a wide range of action-potential-driven exocytotic loads. Our results indicate that when either 5% or 25% of the vesicle pool is used, vesicles are recovered with a half-time on the order of 20 s (24 degrees C). This endocytosis rate was not influenced by operations designed to alter intracellular Ca2+ during membrane retrieval, suggesting that residual Ca2+ after strong stimuli probably does not greatly retard endocytosis. Finally, we have shown that vesicle-destaining kinetics are not strongly influenced by the substantially differing rates at which two marker dyes tested dissociate from membranes. This observation suggests that vesicles remain open long enough for essentially complete dissociation of even the slower dye (a few seconds) or, alternatively, that both dyes readily escape vesicle membrane by lateral diffusion through any exocytotic opening. These data seem most consistent with applicability of the slow-endocytosis, complete-fusion model at low as well as high levels of exocytosis.

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.