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PubMed2001

Aptamer inhibits degradation of platelet proteolytically activatable receptor, PAR-1, by thrombin.

We investigated the in vitro effects of the site-directed thrombin inhibitor-a single-stranded oligonucleotide aptamer (GGTTGGTGTGGTTGG)-on thrombin proteolytic activity towards its two natural substrates: fibrinogen and platelet thrombin receptor (PAR-1). The thrombin aptamer was shown to strongly affect thrombin clotting activity at nanomolar concentrations and thrombin-dependent degradation of proteolytically activatable receptor, PAR-1, exposed on platelet surface membrane at micromolar concentrations. The incubation of PPP with thrombin in the presence of 100-1000 nM aptamer resulted in the significant concentration-dependent prolongation of thrombin time (up to fourfold, P<.0001). Aptamer significantly reduced the thrombin-induced platelet degranulation (46+/-20% inhibition at 0.15 U/ml thrombin, P<.001), as well as thrombin-mediated platelet aggregation in PRP (7+/-10% inhibition at 1 U/ml thrombin, P<.05). Furthermore, aptamer inhibited the thrombin-catalysed cleavage of PAR-1 in a dose-dependent manner, i.e., by 17%, 27% and 70%, respectively, for the concentrations of 100, 500 and 1000 nM (P<.025 by randomised block analysis; P(regression slope)<.0001). We conclude that aptamer is able to considerably attenuate thrombin proteolytic activity regardless of the molecular size of thrombin substrates. Our observations directly proved that aptamer may be successfully used for the inhibition of thrombin activity towards various physiological targets: one related to fibrin generation in the final stage of coagulation cascade, and another concerning the interaction of thrombin with its surface membrane receptor, PAR-1, in blood platelets.

Adult↗
PubMed2000

Capillary electrophoresis separations on a planar chip with the column-coupling configuration of the separation channels

Some basic aspects of capillary electrophoresis (CE) separations on a poly(methyl methacrylate) chip provided with two separation channels in the column-coupling (CC) configuration and on-column conductivity detectors were studied. The CE methods employed in this study included isotachophoresis (ITP), capillary zone electrophoresis (CZE), and CZE with on-line ITP sample pretreatment (ITP-CZE). Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the chip were suppressed, and electrophoresis was a dominant transport process in the separations performed by these methods. Very reproducible migration velocities of the separated constituents were typical under such transport conditions, and consequently, test analytes could be quantified by various ITP techniques with 1-2% RSD. The CC configuration of the separation channels provides means for an effective combination of an enhanced load capacity of the separation system with high detection sensitivities for the analytes in concentration-cascade ITP separations. In this way, for example, succinate, acetate, and benzoate could be separated also in instances when they were present in the loaded sample (1.2 microL) at 1 mmol/L concentrations while their limits of detection ranged from 8 to 12 micromol/L concentrations. A well-defined ITP concentration of the analyte(s) combined with an in-column sample cleanup (via an electrophoretically driven removal of the matrix constituents from the separation compartment) can be integrated into the separations performed on the CC chip. These sample pretreatment capabilities were investigated in ITP-CZE separations of model samples in which nitrite, phosphate, and fluoride (each at a 10 micromol/L concentration) accompanied matrix constituents (sulfate and chloride) at considerably higher concentrations. Here, both the concentration of the analytes and cleanup of the sample were included in the ITP separation in the first separation channel while the second separation channel served for the CZE separation of the ITP pretreated sample and the detection of the analytes.

Journal Article↗
PubMed2003

Therapeutic approaches to the modulation of apoptosis.

The appreciation of the role of apoptosis in the vast majority of diseases affecting humans has revolutionized the discovery and development of drugs targeting inflammation and oncology. Novel therapeutic approaches to modulate disease by regulating apoptosis are currently being tested in preclinical and clinical settings. Enthusiasm for some of these therapies is reflected in the fact that they have received U.S. Food and Drug Administration approval in record time. Approaches include the traditional use of small molecules to target specific players in the apoptosis cascade. They also include radical new approaches such as using antisense molecules to inhibit production of the Bcl-2 protein or antibodies that ligate either death receptors, such as TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), or the MHC (HLA-DR), resulting in the initiation of apoptosis of target cells. Antibodies targeting cell-specific antigens are being used in conjunction with radioactive isotopes to deliver a more specific chemotherapy, particularly in the case of B-cell lymphomas. Other therapies target mitochondria, a key organelle in the apoptosis cascade. This diverse range of therapies includes photodynamic therapy, retinoic acid and arsenic trioxide, all of which induce apoptosis by generating reactive oxygen species. As our understanding of apoptosis increases, further opportunities will arise for tailor-made therapies that will result in improved clinical outcome without the devastating side effects of current interventions.

Animals↗
PubMed2001

Lignan and phenylpropanoid glycosides from Phillyrea latifolia and their in vitro anti-inflammatory activity.

Three phenylpropanoid glycosides (salidroside, syringin and coniferin) and one lignan (phillyrin) isolated from the leaves of Phillyrea latifolia L. (Oleaceae) were tested for interactions with the cyclo-oxygenase and 5-lipoxygenase pathways of arachidonate metabolism in calcium-stimulated mouse peritoneal macrophages and human platelets, and for their effects on cell viability. These compounds are capable of exerting inhibitory actions on enzymes of the arachidonate cascade. Phillyrin, salidroside and syringin exert a preferential effect on the cyclo-oxygenase pathway, inhibiting release of the cyclo-oxygenase metabolites prostaglandin E2 (IC50 values 45.6 microM, 72.1 microM and 35.5 microM, respectively) and to a lesser extent reducing thromboxane B2 levels (IC50 values 168 microM, 154 microM and 29.3 microM, respectively). In contrast, coniferin can be classified as a "dual inhibitor", since it produces reduction in generation of both cyclo-oxygenase (IC50 values 75.2 microM for prostaglandin E2 and 619 microM for thromboxane B2) and 5-lipoxygenase metabolites, but the effects are greater against leukotriene C4 (IC50 value 63.6 microM). Structure-activity relationships of the three phenylpropanoid glycosides are discussed. Thus, like some other compounds found in medicinal herbs, our molecules possess an array of potentially beneficial anti-eicosanoid properties which may, alongside other constituents, contribute to the claimed therapeutic properties of the plant from which they are derived.

Animals↗
PubMed2004

Entry port selection for detecting particle size differences in metered dose inhaler formulations using cascade impaction.

Different sized glass entry ports were evaluated for their drug collection efficiency during aerodynamic particle sizing of metered dose inhalers (MDIs) using cascade impaction. A comparison was made between collection efficiency in the entry port, impactor plates, and filter using the 1 L, 2 L, and 20 L glass entry ports and the USP and twin impinger entry ports. Entry port losses were dependent on the size of entry port selected, with 1-2 L ports showing optimal recovery on impactor plates, compared to the USP entry port. The 1 L entry port was further compared with the USP entry port in its ability to discriminate between subtle changes in particle size distribution (PSD) in an investigational hydrofluoroalkane (HFA)-based MDI formulation. Deliberately induced differences during product manufacture were easily detected using the 1 L entry port with the Andersen cascade impactor. The USP port was unable to distinguish among products with small particle size differences. An alternative entry port such as the 1 L glass entry port used in this study may provide better means of characterizing the PSD during formulation development and stability testing of MDIs.

Aerosols↗
PubMed2006

BcSAK1, a stress-activated mitogen-activated protein kinase, is involved in vegetative differentiation and pathogenicity in Botrytis cinerea.

The gene bcsak1, encoding a mitogen-activated protein kinase (MAPK) of Botrytis cinerea, was cloned and characterized. The protein has high homology to the yeast Hog1 and to corresponding MAPKs from filamentous fungi, but it shows unique functional features. The protein is phosphorylated under osmotic stress, specific fungicides, and oxidative stress mediated by H(2)O(2) and menadione. Northern blot analyses indicate that only a subset of typical oxidative stress response genes is regulated by BcSAK1. In contrast to most other fungal systems, Deltabcsak1 mutants are significantly impaired in vegetative and pathogenic development: they are blocked in conidia formation, show increased sclerotial development, and are unable to penetrate unwounded plant tissue. These data indicate that in B. cinerea the stress-activated MAPK cascade is involved in essential differentiation programs.

Antifibrinolytic Agents↗
PubMed1991

Expression of the herpes simplex virus 1 alpha transinducing factor (VP16) does not induce reactivation of latent virus or prevent the establishment of latency in mice.

A feature of the cascade regulation of herpes simplex virus 1 gene expression in productive infection is that the first genes to be expressed, the alpha genes, are transactivated by a structural component of the virion designated as the alpha transinducing factor (alpha TIF). In this study, we have tested the hypothesis that latent infection of sensory neurons results from the failure of alpha TIF, a tegument protein, to be transported from the nerve endings to the nucleus of the sensory neuron. Two viruses were constructed. The first recombinant virus (R6003) contained a second copy of the alpha TIF gene placed under the control of a metallothionein promoter. The second recombinant virus (R6004) is identical to R6003 except for the presence of a stop codon inserted at amino acid 70 of the second alpha TIF gene. The metallothionein promoter inserted into the viral genome was shown to be expressed, and alpha TIF mRNA was detected by in situ hybridization of sections of trigeminal ganglia of mice infected with R6003, both untreated and those given cadmium injections. In all experiments, there were no significant differences in the recovery of latent virus from mice infected with R6003 or R6004, whether injected with cadmium or not. Cadmium administration at the time of infection and at intervals thereafter did not preclude establishment of latency. In another series of experiments, transgenic mice expressing the metallothionein-driven alpha TIF did not differ from nontransgenic siblings with respect to the incidence of latent virus in trigeminal ganglia. We conclude that the absence of alpha TIF cannot alone account for the establishment of latency.

Animals↗
PubMed1996

Physiology and biochemistry of Drosophila learning mutants.

Single gene mutants of Drosophila that are defective in learning/memory processes have increased substantially our understanding of the physiology, biochemistry, and anatomy underlying conditioned behaviors. Drosophila learning mutants can be separated into two general classes, those with structural defects in the brain and those without (conditioning mutants) any obvious brain alterations. From studies of brain structural mutants, two neuroanatomic areas have merged as important for normal conditioned behavior: the mushroom bodies and the central complex. Biochemical and molecular genetic studies of the conditioning mutants have implicated numerous types of molecules in learning, but the adenosine 3',5'-cyclic monophosphate (cAMP) second messenger pathway has emerged as especially important. Five different genes in this pathway, amnesiac (a product similar to adenylate cyclase activating peptides), dunce (cAMP phosophodiesterase), rutabaga (adenylyl cyclase), DCO (protein kinase A), and dCREB2 (cAMP-response element binding protein), have proven important for normal learning. The products of many of these learning mutants are enriched in mushroom bodies, which highlight the importance of mushroom bodies for normal learning and the cAMP second messenger cascade for the physiology of mushroom body cells in their role(s) underlying learning. Physiological studies of the mutants have demonstrated that plastic properties of synaptic transmission, including facilitation and posttetanic potentiation, are abnormal in the mutants. An appendix describing the currently used paradigms to test Drosophila behavior is included.

Animals↗
PubMed2004

Development of a size-dependent aerosol deposition model utilising human airway epithelial cells for evaluating aerosol drug delivery.

Aerosol delivery to the airways of the human respiratory tract, followed by absorption, constitutes an alternative route of administration for compounds unsuitable for delivery by conventional oral and parenteral routes. The target for aerosol drug delivery is the airways epithelium, i.e. tracheal, bronchial, bronchiolar and alveolar cells, which become the site of drug deposition. These epithelial layers also serve as a barrier to the penetration of inhaled material. An in vitro model for aerosol deposition and transport across epithelia in the human airways may be a good predictor of in vivo disposition. The present preliminary studies begin an investigation that blends the dynamics of aerosol delivery and the basis of an in vitro simulated lung model to evaluate the transport properties of a series of molecular weight marker compounds across human-derived bronchiolar epithelial cell monolayers. An Andersen viable cascade impactor was used as a delivery apparatus for the deposition of size-segregated particles onto monolayers of small airway epithelial cells and Calu-3 cells. It was shown that these cell layers can withstand placement in the impactor, and that permeability can be tested subsequent to removal from the impactor.

Administration, Inhalation↗
PubMed1997

Increased focal adhesion kinase- and urokinase-type plasminogen activator receptor-associated cell signaling in endothelial cells exposed to asbestos.

Exposure of low-passage endothelial cells in culture to nonlethal amounts of asbestos, but not refractory ceramic fiber-1, increases cell motility and gene expression. These changes may be initiated by the fibers mimicking matrix proteins as ligands for receptors on the cell surface. In the present study, 1- to 3-hr exposures of endothelial cells to 5 mg/cm2 of chrysotile asbestos caused marked cell elongation and motility. However, little morphological change was seen when chrysotile was added to cells pretreated with either mannosamine to prevent assembly of glycophosphatidylinositol (GPI)-anchored receptors or with herbimycin A to inhibit tyrosine kinase activity. Affinity purification of GPI-anchored urokinase-type plasminogen activator receptor (uPAR) from chrysotile-exposed cells demonstrated that asbestos altered the profile of proteins and phosphoproteins complexed with this receptor. Tyrosine kinase activities in the complexes were also increased by asbestos. Immunoprecipitations with selective monoclonal antibodies demonstrated that both chrysotile and crocidolite asbestos increase kinase activities associated with p60 Src or p120 focal adhesion kinase (FAK). Further, chrysotile also changed the profile of proteins and phosphoproteins associated with FAK in intact cells. These data suggest that asbestos initiates endothelial cell phenotypic change through interactions with uPAR-containing complexes and that this change is mediated through tyrosine kinase cascades.

Animals↗
PubMed1998

[Rbf1 (RPG-box binding factor), a transcription factor involved in yeast-hyphal transition of Candida albicans].

The major fungal pathogen for fungal diseases which have become a major medical problem in the last few years is Candida albicans, which can grow both in yeast and hyphae forms. This ability of C. albicans is thought to contribute to its colonization and dissemination within host tissues. In a recent few years, accompanying the introduction of molecular biological tools into C. albicans organism, several factors involved in the signal transduction pathway for yeast-hyphal transition have been identified. One MAP kinase pathway in C. albicans, similar to that leading to STE12 activation in Saccharomyces cerevisiae, has been reported. C. albicans strains mutant in these genes show retarded filamentous growth on a solid media but no impairment of filamentous growth in mice. These results suggest two scenarios that a kinase signaling cascade plays a part in stimulating the morphological transition in C. albicans, and that there would be another signaling pathway effective in animals. In this latter true hyphal pathway, although some candidate proteins, such as Efg1 (transcription factor), Int1 (integrin-like membrane protein), or Phr1 (pH-regulated membrane protein), have been identified, it is still too early to say that we understand the whole picture of that cascade. We have cloned a C. albicans gene encoding a novel DNA binding protein, Rbf1, that predominantly localizes in the nucleus, and shows transcriptional activation capability. Disruption of the functional RBF1 genes of C. albicans induced the filamentous growth on all solid and liquid media tested, suggesting that Rbf1 might be another candidate for the true hyphal pathway. Relationships with other factors described above, and the target (regulated) genes of Rbf1 is under investigation.

Candida albicans↗
PubMed2000

Changes in signal transduction in the platelets of patients with peripheral occlusive arterial disease.

BACKGROUND: The finding that platelets of patients with peripheral occlusive arterial disease (POAD) circulate in an activated state prompted us to study platelet signal transduction. We hypothesised that platelet hyperreactivity is caused by changes in intracellular signalling. METHODS EXPERIMENTAL DESIGN: a single blood sample was taken from the antecubital vein of each participant prior to the start of intravenous treatment with prostaglandins. SETTING: patients were recruited from our inpatient Department of Cardiology and Angiology at the University Hospital. PARTICIPANTS: 15 hospitalised patients with symptomatic POAD were randomly selected. Patients receiving antiplatelet drugs and those with diabetes were excluded. The control group consisted of 15 healthy volunteers from the medical staff. INTERVENTIONS: blood tests were performed on the day of admission before any therapeutic intervention. MEASURES: the platelet activation marker P-selectin was quantified on peripheral blood platelets before and after in vitro stimulation with platelet agonists (adenosine diphosphate, thrombin receptor activator peptide-6). The signal transduction cascade was also selectively blocked by preincubation with either: 1) forskolin, 2) phospholipase C inhibitor U-73122, or 3) bisindolylmaleimide. RESULTS: A stronger inhibitory effect on ADP-stimulated platelets was seen in patients with U-73122, as indicated by a decrease in mean fluorescence intensity of 51% versus 34% in controls (p<0.0005). CONCLUSIONS: Our findings support the assumption that changes in platelet signal transduction in POAD lead to platelet hyper-reactivity.

Adenosine Diphosphate↗
PubMedDate not supplied

[Role of calcium and magnesium ions in cerebrospinal fluid in alcoholic-traumatic coma].

The goal of investigation was to determine the role of calcium and magnesium ions in the cerebrospinal fluid in ethylic-traumatic coma. We measured the level of calcium in the cerebrospinal fluid within simple photometric test and the magnesium level within xylidyl blue photometric test. We found a high mortality in patients with high level of calcium in cerebrospinal fluid and low level of magnesium in cerebrospinal fluid. At patients with ethylic-traumatic coma high levels of calcium in cerebrospinal fluid are caused by the excitatory amino acids cascade and increased of hematoencephalic barrier permeability. Decreased levels of magnesium in cerebrospinal fluid are associated with convulsions and a poor prognosis of the patients. These analyses are very important for establishment of prognosis in patients with ethylic-traumatic coma.

Adult↗
PubMed2005

[Experiences with the eradication of the bovine herpes virus 1 in Lower Saxony].

In this paper first the general cascade of animal disease eradication from the voluntary control programme up to culling of infected animals is described. Afterwards the development of the eradication of Bovine Herpes Virus in Lower Saxony is illustrated. Obvious the number of farms which are not eradicating decreases since the testing on BHV1 is obligate in Germany. In 2004 60% of the cattle holdings were free from BHV1. At least the measures which will be taken in Lower Saxony to increase the success of eradication are explained.

Animals↗
PubMed1997

Induction of interleukin-1 and subsequent tissue factor expression by anti-proteinase 3 antibodies in human umbilical vein endothelial cells.

OBJECTIVE: To assess the ability of anti-proteinase 3 (anti-PR3) classic antineutrophil cytoplasmic antibodies (cANCA) to stimulate endothelial expression of tissue factor (TF), which is the main initiator of the coagulation cascade that can lead to endothelial injury and thrombosis in patients with Wegener's granulomatosis. METHODS: Human umbilical vein endothelial cells (HUVEC) were grown to confluence and stimulated with affinity-purified anti-PR3 antibodies, Igs from healthy subjects, and endotoxin (lipopolysaccharide) as positive control. RESULTS: TF activity was generated in anti-PR3-stimulated cells, as shown by a chromogenic test. This activity was inhibited by specific anti-TF antibodies. TF messenger RNA (mRNA) was found in anti-PR3-stimulated cells, as detected by reverse transcriptase-polymerase chain reaction, but not in cells stimulated with irrelevant human Igs or Igs from normal control sera. TF expression reached maximum levels 12 hours after exposure to the anti-PR3 cANCA, and did not require complement. TF mRNA expression was inhibited by cycloheximide, suggesting a requirement for protein synthesis. When added to the incubation medium, interleukin-1 (IL-1) receptor antagonist inhibited the induced TF mRNA expression, suggesting that cANCA-stimulated cells initiate IL-1 synthesis. Moreover, cANCA induced IL-1alpha mRNA before TF mRNA. CONCLUSION: This study showed that anti-PR3 treatment of HUVEC induces sequential expression of IL-1alpha mRNA and TF mRNA, as well as their corresponding proteins. Both proteins could have pathogenic roles in the vasculitic process, since TF is the main initiator of the coagulation cascade.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2002

Extracellular ATP and UTP activate the protein kinase B/Akt cascade via the P2Y(2) purinoceptor in renal mesangial cells.

Extracellular nucleotides can activate a common purinoceptor mediating various cell responses. In this study we report that stimulation of rat mesangial cells with ATP and UTP leads to a rapid activation of the protein kinase B/Akt (PKB) pathway. Time-course studies reveal a rapid and transient phosphorylation of both Ser(473) and Thr(308) of PKB with a maximal effect after 5 min of stimulation. The response is concentration-dependent with a maximal effect at 30 microM of ATP and UTP. Western blot analysis of mesangial cells reveals the expression of the isoenzymes PKB-alpha and PKB-gamma, but not the PKB-beta. ATP and UTP also activate the upstream located PI 3-kinase-dependent kinase. Furthermore, the ATP- and UTP-induced PKB phosphorylation is abolished by two inhibitors of the PI 3-kinase. In addition, suramin, a putative P2Y(2) receptor antagonist, and pertussis toxin, an inhibitor of G(i)/G(o) activation, markedly block ATP- and UTP-induced PKB phosphorylation. A series of ATP and UTP analogues were tested for their ability to stimulate PKB phosphorylation. UTP, ATP and gamma-thio-ATP are the only compounds capable of activating PKB. Stress-induced apoptosis of mesangial cells is reduced by the stable ATP analogue, gamma-thio-ATP, and this inhibitory effect is reversed in the presence of LY 294002. In summary, these results demonstrate that extracellular nucleotides are able to activate the PI 3-kinase/PDK/PKB cascade via the P2Y(2)-receptor and a pertussis toxin-sensitive G(i) protein. Moreover, in mesangial cells this cascade may have an important role in the antiapoptotic response but not in the mitogenic or inflammatory response produced by extracellular nucleotides.

Adenosine Triphosphate↗
PubMed1985

Synthetic lipid A with endotoxic and related biological activities comparable to those of a natural lipid A from an Escherichia coli re-mutant.

A synthetic compound (506), beta (1-6) D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated at 2'-amino and 3'-hydroxyl groups with (R)-3-dodecanoyloxytetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl groups, respectively, and has (R)-3-hydroxytetradecanoyl groups at 2-amino and 3-hydroxyl groups, exhibited full endotoxic activities identical to or sometimes stronger than those of a reference lipid A from an Escherichia coli Re-mutant (strain F515). Endotoxic activities tested include pyrogenicity and leukopenia-inducing activity in rabbits, body weight-decreasing toxicity in normal mice, lethal toxicity in galactosamine-sensitized mice and chicken embryos, and the preparation and provocation of the local Shwartzman reaction in rabbits. Compound 406, a synthetic counterpart of a biosynthetic precursor of lipid A molecule, showed by contrast only weak activities in all of the above assay systems except for the lethality in galactosamine-loaded mice. This finding strongly suggests that the presence of acyloxyacyl groups at the C-2' and C-3' positions of the disaccharide backbone is one of the most important determinant structures of the lipid A molecule for exhibition of strong biological activities characteristic of lipopolysaccharide and its lipid A moiety. The activities of the corresponding 4'-monophosphate (compound 504) and 1-monophosphate (505) analogs were considerably less than those of the parent molecule 506 and the reference F515 lipid A. Regarding other biological activities, not only compound 506 but also compounds 504, 505, and 406 showed definite activities, sometimes comparable to those of F515 lipid A and other reference natural products. These are the activation of Tachypleus tridentatus amoebocyte clotting enzyme cascade and human complement via the classical pathway, mitogenic and polyclonal B-cell activation of murine splenocytes, stimulation of peritoneal macrophages in a guinea pig, enhancement of migration of human blood polymorphonuclear leukocytes, and induction of a serum factor that is cytostatic and cytocidal to L-929 cells in Mycobacterium bovis BCG-primed mice. Relative potencies of test synthetic compounds depended on the assay systems and varied from one system to another. Dephospho-compound 503 lacked most of the biological activities that were definitely observed with phosphorylated compounds, probably because of its insolubility. This study demonstrates the successful chemical synthesis of an E. coli-type lipid A.

Adjuvants, Immunologic↗
PubMed2002

Increased Ras expression and caspase-independent neuroblastoma cell death: possible mechanism of spontaneous neuroblastoma regression.

BACKGROUND: Neuroblastoma undergoes spontaneous regression frequently during its natural course. Although programmed cell death (PCD) has been implicated in this process, accumulating evidence suggests that apoptosis, a form of PCD that is regulated by caspases, may not play a major role. We examined the mechanism(s) of spontaneous regression of neuroblastoma, focusing on the role of Ras, a favorable prognostic marker of neuroblastoma. METHODS: Tumor tissues were analyzed by light microscopy, electron microscopy, and immunohistochemistry to examine cell degeneration and expression of Ras and several indicators of PCD. Cell degeneration was also studied in vitro in neuroblastoma cells transfected with the H-ras gene. All statistical tests were two-sided. RESULTS: Immunohistochemical analyses revealed that Ras expression was increased in areas of cellular degeneration lacking apoptotic characteristics. The degenerating cells were fragmented without nuclear condensation and, essentially, lacked caspase-3 activation and apoptotic DNA fragmentation. These cells had ultrastructural features of autophagic degeneration, another form of PCD that is distinct from apoptosis. Focal areas of degeneration associated with Ras expression were seen more frequently in tumors from patients detected in a mass-screening program (53 [60.9%] of 87) than in tumors from clinically detected, advanced-stage patients over 1 year of age (7 [29.2%] of 24) (P =.006; chi-square test), suggesting a positive relationship between Ras-associated degeneration and probability of spontaneous regression/favorable prognosis. The characteristic features of Ras-associated nonapoptotic degeneration observed in tumor samples were recapitulated in vitro by transfection-mediated Ras expression, and Ras-mediated degeneration was augmented by TrkA, another favorable prognostic marker. CONCLUSIONS: High-level expression of H-Ras in neuroblastoma cells is associated with caspase cascade-independent, nonapoptotic PCD. This Ras-mediated nonapoptotic tumor cell death may play a key role in spontaneous regression of neuroblastoma.

Apoptosis↗