Cerebral neoplasms in L-2 hydroxyglutaric aciduria: 3 new cases and meta-analysis of literature data.

SUMMARY: Increasing evidence suggests that patients with L2-HGA have a predisposition to cerebral neoplasms. This may be related to the pathologic accumulation of L2-HG because high amounts of 2-HG have been found in brain neoplasms that have IDH1 mutations. Our experience, on the basis of 11 previously unreported cases of L2-HGA, 3 of which developed cerebral neoplasms during the course of the disease, also supports an association between L2-HGA and cerebral neoplasms. We conducted a meta-analysis of published data, and we identified 295 patients (including our 11 patients) with L2-HGA. In 14 patients, the metabolic disorder was associated with cerebral neoplasms, suggesting an approximately 5% prevalence rate of CNS neoplasms in patients with L2-HGA; nonetheless, it may still be an underestimate. L2-HGA is an important disease "model" that provides further evidence to support the recently proposed pathogenetic role of 2-HG in the development of cerebral neoplasms.

A molecular profile of the mouse gastric parietal cell with and without exposure to Helicobacter pylori.

The parietal cell (PC) plays an important role in normal gastric physiology and in common diseases of the stomach. Although the genes involved in acid secretion are well known, there is limited molecular information about other aspects of PC function. We have generated a comprehensive database of genes expressed preferentially in PCs relative to other gastric mucosal cell lineages. PCs were purified from FVB/N mouse stomachs by lectin panning. cRNA generated from PC-enriched (PC(+)) and PC-depleted (PC(-)) populations were used to query oligonucleotide-based microarrays. False-positive signals were filtered by using a new algorithm for noise reduction and selected results independently audited by real-time quantitative reverse transcription (RT)-PCR. The annotated database of 240 genes reveals previously unappreciated aspects of cellular function, including factors that may mediate PC regulation of gastric stem cell proliferation. PC(+) and PC(-) expression profiles were also prepared from germ-free mice 2 and 8 weeks after colonization with a clinical isolate of Helicobacter pylori (Hp)--the pathogen that produces acid-peptic disease (gastritis, ulcers) in humans. Whereas PC(+) gene expression was remarkably constant, the PC(-) fractions demonstrated a robust, evolving host response, with increased expression of genes involved in cell motility/migration, extracellular matrix interactions, and IFN responses. The consistency of PC(+) gene expression allowed identification of a cohort of 92 genes enriched in PCs under all conditions studied. These genes provide a molecular profile that can be used to define this epithelial lineage under a variety of physiologic, pharmacologic, and pathologic stimuli.

DNA microarrays and beyond: completing the journey from tissue to cell.

For the cell biologist, identifying changes in gene expression using DNA microarrays is just the start of a long journey from tissue to cell. We discuss how chip users can first filter noise (false-positives) from daunting microarray datasets. Combining laser capture microdissection with real-time polymerase chain reaction and reverse transcription is a helpful follow-up step that allows expression of selected genes to be quantified using sensitive new in situ hybridization and immunohistochemical methods based on tyramide signal amplification.

A new approach for filtering noise from high-density oligonucleotide microarray datasets.

Although DNA microarrays are powerful tools for profiling gene expression, the dynamic range and the sheer number of signals produced require efficient procedures for distinguishing false positive results (noise) from changes in expression that are 'real' (independently reproducible). We have developed an approach to filter noise from datasets generated when high density oligonucleotide-based microarrays are used to compare two distinct RNA populations. First, we performed comparisons between chips hybridized with cRNAs prepared from an identical starting RNA population; an 'Increase' or 'Decrease' call in such a comparison was defined as a false positive. Plotting the average distribution of these false positive signal intensities across 18 such comparisons of nine independent RNA preparations allowed us to develop a series of noise-filtering look-up tables (LUTs). Using a database of 70 separate chip-to-chip comparisons between distinct RNA preparations prepared by different workers at different sites and at different times, we show that the LUTs can be used to predict the likelihood that a given transcript called Increased or Decreased in one comparison will again be called Increased or Decreased in a replicate comparison. Evidence is presented that this LUT-based scoring system provides greater predictive value for reproducible microarray results than imposition of arbitrary fold-change thresholds and accurately predicts which microarray-identified changes will be validated by independent assays such as quantitative real-time PCR.

The endodontic autopsy: a valid learning tool.

Clinical success rates in endodontics can be improved if the causes of failures can be determined, prevented, or corrected. The endodontic autopsy is recommended as a method for determining preoperative, operative, and postoperative causes of failures in endodontics.

Biological basis for clinical success: pulp protection and the tooth-restoration interface.

This article provides biological and technological information that strengthens clinicians' understanding of cohesive hybridization and pulp therapy in order to support their routine use of bonding and resin systems. Utilizing cohesive systems, clinicians should experience several advantages over traditional water-soluble base and liner systems. When properly applied, cohesive hybridization of vital dentin prevents immediate postoperative hypersensitivity under all restorations and completely seals the entire tooth-restoration interface, which provides a reduction in recurrent caries.

Activation of a PP2A-like phosphatase and dephosphorylation of tau protein characterize onset of the execution phase of apoptosis.

The execution phase is an evolutionarily conserved stage of apoptosis that occurs with remarkable temporal and morphological uniformity in most if not all cell types regardless of the condition used to induce death. Characteristic features of apoptosis such as membrane blebbing, DNA fragmentation, chromatin condensation, and cell shrinkage occur during the execution phase; therefore, there is considerable interest in defining biochemical changes and signaling events early in the execution phase. Since onset of the execution phase is asynchronous across a population with only a small fraction of cells in this stage at any given time, characterizing underlying biochemical changes is difficult. An additional complication is recent evidence suggesting that the execution phase occurs after cells commit to die; thus, agents that modulate events in the execution phase may alter the morphological progression of apoptosis but will not affect the time-course of death. In the present study, we use a single cell approach to study and temporally order biochemical and cytoskeletal events that occur specifically in the execution phase. Microtubules de-acetylate and disassemble as terminally differentiated PC12 cells enter the execution phase following removal of nerve growth factor. Using phosphorylation sensitive antibodies to tau, we show that this microtubule-stabilizing protein becomes dephosphorylated near the onset of the execution phase. Low concentrations of okadaic acid inhibit dephosphorylation suggesting a PP2A-like phosphatase is responsible. Transfecting (tau) into CHO cells to act as a 'reporter' protein shows a similar dephosphorylation of (tau) by a PP2A-like phosphatase during the execution phase following induction of apoptosis with UV irradiation. Therefore, activation of PP2A phosphatase occurs at the onset of the execution phase in two very different cell types following different initiators of apoptosis which is consistent with activation of PP2A phosphatase being a common feature of the execution phase of apoptosis. Experiments using either taxol to inhibit microtubule disassembly or okadaic acid to inhibit tau dephosphorylation suggest that microtubule disassembly is necessary for tau dephosphorylation to occur. Thus, we propose that an early step in the execution phase (soon after a cell commits to die) is microtubule disassembly which frees or activates PP2A to dephosphorylate tau as well as other substrates.

Apoptotic membrane blebbing is regulated by myosin light chain phosphorylation.

The evolutionarily conserved execution phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the execution phase of apoptosis.

Differentiation to an NGF-dependent state and apoptosis following NGF removal both occur asynchronously in cultures of PC12 cells.

Long-term timelapse videomicroscopy was used to investigate the relationships and transitions between mitosis, differentiation, and apoptosis in cultures of NGF-differentiated PC12 cells. After 4 days in NGF, cultures were at an early stage of neuronal differentiation. Removal of NGF led to an appreciable increase in apoptosis with no effect on the relatively high mitotic rate. After 7 days in NGF, cells were more neuronal; NGF withdrawal again resulted in no change in the low mitotic rate but an even greater increase in apoptosis, eventually leading to considerable net loss of cells. After 10 days, cells were terminally differentiated; removal of NGF did not affect the negligible mitotic rate but induced a dramatic increase in apoptosis resulting in death of most of the cells. Apoptosis in the fraction of cells that had become NGF-dependent followed a similar timecourse and was characterized by the same morphology at all three differentiation states. Thus, acquisition of NGF-dependence in PC12 cultures seemed to be the result of a steadily increasing percentage of cells that had each undergone a relatively rapid transition to a postmitotic, NGF-sensitive state. These studies were also helpful for elucidating the timing of apoptosis. Onset of apoptosis was markedly asynchronous within a culture, but the active, blebbing phase, once initiated, always lasted about 45 min, regardless of differentiation state or time spent without NGF. Thus, the active phase might represent a conserved sequence of events that every cell must ultimately undergo before apoptotic death.

Development of polarity in human erythroleukemia cells: roles of membrane ruffling and the centrosome.

Cultured human erythroleukemia (HEL) cells were used to study the genesis of polarity in single cells. HEL cells grow in suspension in culture medium, but attach and spread on fibronectin when treated with 10 nM phorbol myristate acetate. If the spread cells are treated with dibutyryl cyclic adenosine monophosphate, about 50% of the cells polarize and form very striking elongated processes. Time-lapse video microscopy showed that elongation develops in these cells because the anterior pole of the cell, which bears a small ruffled membrane, moves slowly (approximately 0.16 microgram/min) forward on the substratum elongating the posterior pole or tail behind it. Using indirect immunofluorescence we found that elongation of the tail correlates with the development of long microtubule bundles emanating from the centrosome, which is located posterior to the nucleus on the trailing side of the cell. Incubation with nocodazole, which inhibited development of the long microtubules and the elongation, resulted in a centrosome positioned over the nucleus in 45% of the cells and extension of the membrane ruffling to many points around the cell's periphery. Unexpectedly, time-lapse video microscopy demonstrated that the treated cultures also contained some smaller cells with very marked anterior ruffles and short tails. These cells moved rapidly about the culture dish (maximum 0.8 microgram/min; average 0.5 microgram/min). In these fast moving cells the centrosome was also located posterior to the nucleus. Several recent reports have stressed the importance of relocation of the centrosome to an anterior position in cells developing polarity after experimental wounding. Our results show that both striking polarization and rapid motility can occur without such a relocation. The polarity induced in the HEL cells correlates most clearly with the limitation of membrane ruffling to one region; this limitation is removed by microtubule disassembly. We therefore propose that localized ruffling is the critical first step in polarized motility generally, and that centrosomal position is related to other factors.

Surgical correction of medial subluxation of the patella.

We evaluated the results of a surgical procedure to correct medial subluxation of the patella in 63 patients (65 knees), most of whom had undergone a lateral retinacular release. We performed a direct repair or a reconstruction of the lateral patellotibial ligament using locally available tissue such as strips of iliotibial band or patellar tendon. Followup averaged 53.7 months (range, 24 to 99). Outcome was based on the examiner's inability to clinically reproduce the patient's painful medial subluxation and on the patient's general impression of his or her improved functional status. Forty-four patients (68%) reported improvement in their functional levels and 49 (75%) reported that they were subjectively improved by the procedure. Overall, 50 patients (80%) had a rating of good or excellent. Six knees required a second surgical reconstruction because of failure to improve or because of a reinjury. Analysis of overall clinical outcome revealed no significant relationships based on the patient's age at the time of the initial procedure, sex, or length of followup (P > 0.10). Reconstitution of the lateral patellotibial ligament effectively corrected medial subluxation of the patella and long-term results of this salvage procedure were satisfactory.

Metabolic and energetic changes during apoptosis in neural cells.

Changes in cellular energetic and metabolic parameters were analyzed at several time points during apoptosis of differentiated PC12 cells following removal of nerve growth factor (NGF). As approximately 60% of the population died during the period of study (24 h), most of the measured metabolic indicators declined over time. However, this decline paralleled the overall decrease in cellular viability, suggesting that, in individual cells, a compromised metabolic state occurred suddenly and very late in the death process. For example, when expressed as a function of viable cells, protein and RNA synthesis did not decrease until 24 h. Glucose utilization in live cells was never significantly reduced relative to control levels; lactate production decreased slightly within 4-8 h after NGF removal, but eventually rebounded to 122% of control levels by 24 h. ATP levels dropped 27% in an early predeath period, but then returned to near control levels (on a per-live-cell basis) once the population actively began to die. The ATP/ADP ratio remained at least 84% of control throughout. UTP/UDP and GTP/GDP ratios did not change significantly at any time point.

Neuronal cell death: searching for the smoking gun.

During the past year,several model systems have been developed to identify cellular and biochemical events involved in neuronal cell death, to investigate the role of bcl-2 in cell survival, and to characterize the relationship between cell death and the cell cycle.

A system for characterizing cellular and molecular events in programmed neuronal cell death.

A model system has been established in which PC12 cells are converted to neuronal-like cells that undergo transcription-dependent cell death following removal of NGF. Nineteen sublines of PC12 cells were tested to establish parameters for making cells dependent on NGF for survival. In most sublines, a relatively small percentage of cells become dependent on NGF for survival, and following removal of NGF, most of the cells begin proliferating in serum-containing medium. In several sublines, however, a significant percentage of cells die following removal of NGF. One of these sublines, PC6-3, can be grown under conditions in which 90% of the cells undergo transcription-dependent cell death following removal of NGF in either serum-free or serum-containing medium. Fourteen hours after removing NGF, 50% of the cells are committed to die, while initial morphological signs of cell death as determined by time-lapse videomicroscopy occur 2-6 hr later and include loss of neurites followed by a 1-3 hr period of active membrane "blebbing" and protrusions. Cell death can be blocked by the RNA synthesis inhibitor actinomycin D, the protein synthesis inhibitor cycloheximide, KCl, basic fibroblast growth factor, or dibutryl-cAMP, but not by epidermal growth factor, leupeptin, or the endonuclease inhibitor aurintricarboxylic acid (ATA). Removal of NGF activates an endonuclease that causes nucleosomal laddering of the DNA; however, endonuclease activity does not appear to be required for cell death. In agreement with previous studies (Batistatou and Greene, 1991; Rukenstein et al., 1991) demonstrating that naive PC12 cells undergo transcription-independent cell death when shifted into serum-free medium in the absence of growth factors, all cell lines tested except for one die when cultured in RPMI medium lacking growth factors. DNA fragmentation is a prominent feature of transcription-independent cell death, and death can be blocked with NGF, ATA, and dibutryl-cAMP but not with actinomycin D or KCl. The PC12 model system described here should be useful for identifying cell death genes and for characterizing cellular and molecular events in programmed neuronal cell death.

Tongue in cheek? Or in the bottle?

The case of an eight-year-old girl whose tongue became entrapped in a bottle is presented. Management options are discussed. Successful management utilizing a positive pressure technique is described and illustrated.