RB-mediated suppression of spontaneous multiple neuroendocrine neoplasia and lung metastases in Rb+/− mice

Alterations in pathways mediated by retinoblastoma susceptibility gene (RB) product are among the most common in human cancer. Mice with a single copy of the Rb gene are shown to develop a syndrome of multiple neuroendocrine neoplasia. The earliest Rb-deficient atypical cells were identified in the intermediate and anterior lobes of the pituitary, the thyroid and parathyroid glands, and the adrenal medulla within the first 3 months of postnatal development. These cells form gross tumors with various degrees of malignancy by postnatal day 350. By age of 380 days, 84% of Rb+/− mice exhibited lung metastases from C-cell thyroid carcinomas. Expression of a human RB transgene in the Rb+/− mice suppressed carcinogenesis in all tissues studied. Of particular clinical relevance, the frequency of lung metastases also was reduced to 12% in Rb+/− mice by repeated i.v. administration of lipid-entrapped, polycation-condensed RB complementary DNA. Thus, in spite of long latency periods during which secondary alterations can accumulate, the initial loss of Rb function remains essential for tumor progression in multiple types of neuroendocrine cells. Restoration of RB function in humans may prove an effective general approach to the treatment of RB-deficient disseminated tumors.

Tumor necrosis factor α plays a central role in immune-mediated clearance of adenoviral vectors

Adenovirus (Ad) gene transfer vectors are rapidly cleared from infected hepatocytes in mice. To determine which effector mechanisms are responsible for elimination of the Ad vectors, we infected mice that were genetically compromised in immune effector pathways [perforin, Fas, or tumor necrosis factor α (TNF-α)] with the Ad vector, Ad5-chloramphenicol acetyl transferase (CAT). Mice were sacrificed at 7–60 days postinfection, and the levels of CAT expression in the liver determined by a quantitative enzymatic assay. When the livers of infected mice were harvested 28 days postinfection, the levels of CAT expression revealed that the effectors most important for the elimination of the Ad vector were TNF-α > Fas > perforin. TNF-α did not have a curative effect on infected hepatocytes, as the administration of TNF-α to infected severe combined immunodeficient mice or to infected cultures in vitro had no specific effect on virus persistence. However, TNF-α-deficient mice demonstrated a striking reduction in the leukocytic infiltration early on in the infection, suggesting that TNF-α deficiency resulted in impaired recruitment of inflammatory cells to the site of inflammation. In addition, the TNF-deficient mice had a significantly reduced humoral immune response to virus infection. These results demonstrate a dominant role of TNF-α in elimination of Ad gene transfer vectors. This result is particularly important because viral proteins that disable TNF-α function have been removed from most Ad vectors, rendering them highly susceptible to TNF-α-mediated elimination.

Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30–100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant ΔF508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.

Spontaneous mutations recovered as mosaics in the mouse specific-locus test

The specific-locus test (SLT) detects new mutants among mice heterozygous for seven recessive visible markers. Spontaneous mutations can be manifested not only as singleton whole-body mutants in controls (for which we report new data), but as mosaics—either visible (manifesting mottled coat color) in the scored generation (G2) or masked, among the wild-type parental generation (G1). Masked G1 mosaics reveal themselves by producing clusters of whole-body mutants in G2. We provide evidence that most, if not all, mosaics detected in the SLT (both radiation and control progenies) result from a single-strand spontaneous mutation subsequent to the last premeiotic mitosis and before the first postmeiotic one of a parental genome—the “perigametic interval.” Such events in the genomes of the G1 and G0 result, respectively, in visible and masked 50:50 mosaics. Per cell cycle, the spontaneous mutation rate in the perigametic interval is much higher than that in pregamete mitotic divisions. A clearly different locus spectrum further supports the hypothesis of different origin, and casts further doubt on the validity of the doubling-dose risk-estimation method. Because mosaics cannot have arisen in mitotic germ cells, and are not induced by radiation exposure in the perigametic interval, they should not be included in calculations of radiation-induced germ-line mutation rates. For per-generation calculations, inclusion of mosaics yields a spontaneous frequency 1.7 times that calculated from singletons alone for mutations contributed by males; including both sexes, the multiple is 2.2.

Prolonged activation of the N-methyl-d-aspartate receptor–Ca2+ transduction pathway causes spontaneous recurrent epileptiform discharges in hippocampal neurons in culture

The molecular basis for developing symptomatic epilepsy (epileptogenesis) remains ill defined. We show here in a well characterized hippocampal culture model of epilepsy that the induction of epileptogenesis is Ca2+-dependent. The concentration of intracellular free Ca2+ ([Ca2+]i) was monitored during the induction of epileptogenesis by prolonged electrographic seizure activity induced through low-Mg2+ treatment by confocal laser-scanning fluorescent microscopy to directly correlate changes in [Ca2+]i with alterations in membrane excitability measured by intracellular recording using whole-cell current–clamp techniques. The induction of long-lasting spontaneous recurrent epileptiform discharges, but not the Mg2+-induced spike discharges, was prevented in low-Ca2+ solutions and was dependent on activation of the N-methyl-d-aspartate (NMDA) receptor. The results provide direct evidence that prolonged activation of the NMDA–Ca2+ transduction pathway causes a long-lasting plasticity change in hippocampal neurons causing increased excitability leading to the occurrence of spontaneous, recurrent epileptiform discharges.

A model for spontaneous B-lineage lymphomas in IgHμ-HOX11 transgenic mice

HOX11, a divergent homeodomain-containing transcription factor, was isolated from the breakpoint of the nonrandom t(10;14)(q24;q11) chromosome translocation found in human T cell acute lymphoblastic leukemias. The translocation places the HOX11 coding sequence under the transcriptional control of TCR α/δ regulatory elements, resulting in ectopic expression of a normal HOX11 protein in thymocytes. To investigate the oncogenic potential of HOX11, we targeted its expression in lymphocytes of transgenic mice by placing the human cellular DNA under the transcriptional control of Ig heavy chain or LCK regulatory sequences. Only IgHμ-HOX11 mice expressing low levels of HOX11 were viable. During their second year of life, all HOX11 transgenic mice became terminally ill with more than 75% developing large cell lymphomas in the spleen, which frequently disseminated to thymus, lymph nodes, and other nonhematopoietic tissues. Lymphoma cells were predominantly clonal IgM+IgD+ mature B cells. Repopulation of severe combined immunodeficient mice with cells from hyperplastic spleens indicated that the HOX11 tumor phenotype was transplantable. Before tumor development, expression of the transgene did not result in perturbations in lymphopoiesis; however, lymphoid hyperplasia involving the splenic marginal zones was present in 20% of spleens. Our studies provide direct evidence that expression of HOX11 in lymphocytes leads to malignant transformation. These mice are a useful model system to study mechanisms involved in transformation from B-lineage hyperplasia to malignant lymphoma and for testing novel approaches to therapy. They represent a novel animal model for non-Hodgkin’s lymphoma of peripheral mature B cell origin.

The prion model for [URE3] of yeast: Spontaneous generation and requirements for propagation

The genetic properties of the non-Mendelian element, [URE3], suggest that it is a prion (infectious protein) form of Ure2p, a mediator of nitrogen regulation in Saccharomyces cerevisiae. Into a ure2Δ strain (necessarily lacking [URE3]), we introduced a plasmid overproducing Ure2p. This induced the frequent “spontaneous generation” of [URE3], with properties identical to the original [URE3]. Altering the translational frame only in the prion-inducing domain of URE2 shows that it is Ure2 protein (and not URE2 RNA) that induces appearance of [URE3]. The proteinase K-resistance of Ure2p is unique to [URE3] strains and is not seen in nitrogen regulation of normal strains. The prion-inducing domain of Ure2p (residues 1–65) can propagate [URE3] in the absence of the C-terminal part of the molecule. In contrast, the C-terminal part of Ure2p cannot be converted to the prion (inactive) form without the prion-inducing domain covalently attached. These experiments support the prion model for [URE3] and extend our understanding of its propagation.

A Molecular Network That Produces Spontaneous Oscillations in Excitable Cells of Dictyostelium

A network of interacting proteins has been found that can account for the spontaneous oscillations in adenylyl cyclase activity that are observed in homogenous populations of Dictyostelium cells 4 h after the initiation of development. Previous biochemical assays have shown that when extracellular adenosine 3′,5′-cyclic monophosphate (cAMP) binds to the surface receptor CAR1, adenylyl cyclase and the MAP kinase ERK2 are transiently activated. A rise in the internal concentration of cAMP activates protein kinase A such that it inhibits ERK2 and leads to a loss-of-ligand binding by CAR1. ERK2 phosphorylates the cAMP phosphodiesterase REG A that reduces the internal concentration of cAMP. A secreted phosphodiesterase reduces external cAMP concentrations between pulses. Numerical solutions to a series of nonlinear differential equations describing these activities faithfully account for the observed periodic changes in cAMP. The activity of each of the components is necessary for the network to generate oscillatory behavior; however, the model is robust in that 25-fold changes in the kinetic constants linking the activities have only minor effects on the predicted frequency. Moreover, constant high levels of external cAMP lead to attenuation, whereas a brief pulse of cAMP can advance or delay the phase such that interacting cells become entrained.

Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide

A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant p53 genes, in a L9V/HLA-A2 specific and restricted fashion. Thus, the normal tolerance against endogenously processed p53 protein-derived self-epitopes can be broken by peptide-specific in vitro priming. p53 protein-derived wild-type peptides might thus represent tumor associated target molecules for immunotherapeutical approaches.

Dramatically decreased high density lipoprotein cholesterol, increased remnant clearance, and insulin hypersensitivity in apolipoprotein A-II knockout mice suggest a complex role for apolipoprotein A-II in atherosclerosis susceptibility

Apolipoprotein (apo) A-II is the second most abundant apolipoprotein in high density lipoprotein (HDL). To study its role in lipoprotein metabolism and atherosclerosis susceptibility, apo A-II knockout mice were created. Homozygous knockout mice had 67% and 52% reductions in HDL cholesterol levels in the fasted and fed states, respectively, and HDL particle size was reduced. Metabolic turnover studies revealed the HDL decrease to be due to both decreased HDL cholesterol ester and apo A-I transport rate and increased HDL cholesterol ester and apo A-I fractional catabolic rate. The apo A-II deficiency trait was bred onto the atherosclerosis-prone apo E-deficient background, which resulted in a surprising 66% decrease in cholesterol levels due primarily to decreased atherogenic lipoprotein remnant particles. Metabolic turnover studies indicated increased remnant clearance in the absence of apo A-II. Finally, apo A-II deficiency was associated with lower free fatty acid, glucose, and insulin levels, suggesting an insulin hypersensitivity state. In summary, apo A-II plays a complex role in lipoprotein metabolism, with some antiatherogenic properties such as the maintenance of a stable HDL pool, and other proatherogenic properties such as decreasing clearance of atherogenic lipoprotein remnants and promotion of insulin resistance.

Nitric oxide-related species inhibit evoked neurotransmission but enhance spontaneous miniature synaptic currents in central neuronal cultures

Nitric oxide (NO·) does not react significantly with thiol groups under physiological conditions, whereas a variety of endogenous NO donor molecules facilitate rapid transfer to thiol of nitrosonium ion (NO+, with one less electron than NO·). Here, nitrosonium donors are shown to decrease the efficacy of evoked neurotransmission while increasing the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs). In contrast, pure NO· donors have little effect (displaying at most only a slight increase) on the amplitude of evoked EPSCs and frequency of spontaneous mEPSCs in our preparations. These findings may help explain heretofore paradoxical observations that the NO moiety can either increase, decrease, or have no net effect on synaptic activity in various preparations.

Effects of salicylates and aminoglycosides on spontaneous otoacoustic emissions in the Tokay gecko

The high sensitivity and sharp frequency discrimination of hearing depend on mechanical amplification in the cochlea. To explore the basis of this active process, we examined the pharmacological sensitivity of spontaneous otoacoustic emissions (SOAEs) in a lizard, the Tokay gecko. In a quiet environment, each ear produced a complex but stable pattern of emissions. These SOAEs were reversibly modulated by drugs that affect mammalian otoacoustic emissions, the salicylates and the aminoglycoside antibiotics. The effect of a single i.p. injection of sodium salicylate depended on the initial power of the emissions: ears with strong control SOAEs displayed suppression at all frequencies, whereas those with weak control emissions showed enhancement. Repeated oral administration of acetylsalicylic acid reduced all emissions. Single i.p. doses of gentamicin or kanamycin suppressed SOAEs below 2.6 kHz, while modulating those above 2.6 kHz in either of two ways. For ears whose emission power at 2.6–5.2 kHz encompassed more than half of the total, individual emissions displayed facilitation as great as 35-fold. For the remaining ears, emissions dropped to as little as one-sixth of their initial values. The similarity of the responses of reptilian and mammalian cochleas to pharmacological intervention provides further evidence for a common mechanism of cochlear amplification.

Spontaneous heterosis in larval life-history traits of hemiclonal frog hybrids

European water frog hybrids Rana esculenta (Rana ridibunda × Rana lessonae) reproduce hemiclonally, transmitting only their ridibunda genome to gametes. We compared fitness-related larval life-history traits of natural R. esculenta from Poland with those of the two sympatric parental species and of newly generated F1 hybrids. Compared with either parental species, F1 hybrid offspring had higher survival, higher early growth rates, a more advanced developmental stage by day 49, and earlier metamorphosis, but similar mass at metamorphosis. R. esculenta from natural lineages had trait values intermediate between those of F1 offspring and of the two parental species. The data support earlier observations on natural R. esculenta that had faster larval growth, earlier metamorphosis, and higher resistance to hypoxic conditions compared with either parental species. Observing larval heterosis in F1 hybrids in survival, growth rate, and time to metamorphosis, however, at an even higher degree than in hybrids from natural lineages, demonstrates that heterosis is spontaneous and results from hybridity per se rather than from subsequent interclonal selection; in natural lineages the effects of hybridity and of clonal history are confounded. This is compelling evidence for spontaneous heterosis in hybrid clonals. Results on hemiclonal fish hybrids (Poeciliopsis) showed no spontaneous heterosis; thus, our frog data are not applicable to all hybrid clonals. Our data do show, however, that heterosis is an important potential source for the extensively observed ecological success of hybrid clonals. We suggest that heterosis and interclonal selection together shape fitness of natural R. esculenta lineages.

A role for Src kinase in spontaneous epileptiform activity in the CA3 region of the hippocampus

Members of the Src family of nonreceptor protein tyrosine kinases (PTKs) have been implicated in the regulation of cellular excitability and synaptic plasticity. We have investigated the role of these PTKs in in vitro models of epileptiform activity. Spontaneous epileptiform discharges were induced in vitro in the CA3 region of rat hippocampal slices by superfusion with the potassium channel blocker 4-aminopyridine in Mg2+-free medium. In hippocampal slices treated in this fashion, Src kinase activity was increased and the frequency of epileptiform discharges could be greatly reduced by inhibitor of the Src family of PTKs, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), but not by the inactive structural analog 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also reduced epileptiform activity induced by either 4-aminopyridine or Mg2+-free medium alone. These observations demonstrate a role for Src family PTKs in the pathophysiology of epilepsy and suggest potential therapeutic targets for antiepileptic therapy.

Spontaneous rupture of malarial spleen: two case reports and review of literature

Malaria has long been among the most common diseases in the southeast Anatolia region of Turkey. In 1992, 18676 cases were diagnosed in Turkey, and Diyarbakir city had the highest incidence (4168 cases), followed by SanliUrfa city (3578 cases). Malaria was especially common during 1994 and 1995, with 84 345 and 82 094 cases being diagnosed in these years, respectively. Spontaneous rupture of malarial spleen is rare. We saw two cases during 1998, which are reported herein. Both patients were male, and were receiving chloroquine treatment for an acute attack of malaria. One of the patients had developed abdominal pain and palpitations, followed by fainting. The other patient had abdominal pain and fever. Explorative laparotomy revealed an enlarged spleen in both patients. Splenectomy was performed in both patients. We have identified 15 episodes of spontaneous rupture of the spleen in the English language literature published since 1961. Because of increased travel to endemic areas and resistance to antimalarial drugs, malaria is a major medical problem that is becoming increasingly important to surgeons worldwide. Malaria is a particularly important problem in the southeast Anatolia region of Turkey. Prophylactic precautions should be taken by tourists who travel to this region, especially during the summer.