Analysis of differentially expressed genes discovers Latroeggtoxin VI-induced changes and SYNJ1 as a main target in PC12 cells.

BACKGROUND: Previous preliminary work found that Latroeggtoxin-VI (LETX-VI), a proteinaceous neurotoxin from the eggs of spider Latrodectus tredecimguttatus, could promote the synthesis and release of dopamine in PC12 cells. However, the underlying mechanisms have not been fully clear. Here, the effects of LETX-VI on the gene expression profile and dopamine in PC12 cells were analyzed with the differential transcriptome-based strategies. RESULTS: After treatment of PC12 cells with LETX-VI for 24 h, a total of 356 differentially expressed transcripts were identified. Of them 165 were up-regulated and 191 down-regulated. Relevant GO analysis indicated that LETX-VI modulated the expression of certain genes and thereby affected multiple biological processes in PC12 cells, including protein metabolism, nucleic acid metabolism, substance transport, signaling, neurotransmitter metabolism and release. When western blot analysis was employed to confirm the abundance levels of synaptojanin 1 and synuclein alpha interacting protein, the representatives of highly up- and down-regulated transcript-encoded proteins that are closely related with dopamine respectively, it was found that the level of synaptojanin 1 in the PC12 cells treated with LETX-VI was increased, whereas that of synuclein alpha interacting protein was not obviously altered, suggesting that synaptojanin 1 may be much more involved in the effects of LETX-VI on dopamine. After synaptojanin 1 level was knocked down using siRNA, the levels of both total and released dopamine were significantly decreased, indicating that synaptojanin 1 is a protein positively modulating the synthesis and secretion of dopamine. When the PC12 cells with knocked down synaptojanin 1 were treated by LETX-VI, the adverse effects of synaptojanin 1 knockdown on dopamine were attenuated, confirming that LETX-VI promotes the synthesis and secretion of dopamine at least partially by enhancing the expression of the gene SYNJ1 encoding synaptojanin 1. CONCLUSIONS: This work demonstrates that LETX-VI exerts multiple regulatory effects on the cellular processes in PC12 cells by altering the gene expression profile. LETX-VI modulates the expression of the genes closely related to the synthesis, transport and release of neurotransmitters especially dopamine in PC12 cells, with the gene SYNJ1 encoding synaptojanin 1 as a main target.

Cyto-genotoxic and oxidative effects of a continuous UV-C treatment of liquid egg products.

UV-C treatment of food is a promising non-thermal processing technology to improve food safety and preservation. Most of the chemical constituents of food absorb UV-C light that can lead to chemical modifications and quality changes. This work investigated the effects of UV-C treatment of liquid egg products on lipid, protein oxidations and potential cyto- and genotoxic effects on intestinal epithelial cells in vitro. Egg preparations (egg white, yolk, liquid whole egg) were treated with UV-C (254 nm, volumetric doses between 0 and 115,619 J L(-1)) using a commercial UV-C processing unit equipped with a Dean Flow reactor. UV-C treatment at high doses (from 32,181 J L(-1), about 2 times higher than that needed to inactivate 5 log of relevant microorganisms) showed an increased lipid oxidation in egg yolk and slight effects in liquid whole eggs; this was confirmed by slightly but not statistically significant increased peroxide values. UV-C induced also slight protein damage, characterised by the total sulfhydryl group reduction. These UV-C-induced oxidative modifications in egg preparations however did not cause any increase in the cyto- or genotoxic (DNA strand breaks) effects in intestinal Caco-2 cells.

Mortality and testicular derangements in red flour beetles, Tribolium castaneum (Herbst) exposed to hen's egg white proteins.

Red flour beetle (T. castaneum) is a major pest of stored grains and is known for its adaptability to all classes of insecticides. The present study was carried out to determine the insecticidal potential of egg white proteins to manage beetle population. Protein samples obtained through salt fractionation were lyophilized and were used separately and simultaneously in different concentrations by adding them to wheat flour and milk powder. The results indicated that the mortality rate of the adult beetles was dependent on the type of treatment, concentration of protein samples and duration of feeding. In multiple-choice feeding trials beetles showed their movement towards the control section as the concentration of treatment increases. Marked abnormalities were observed in appearance and dimensions of the testes which indicated that the egg white proteins caused considerable effect on the process of spermatogenesis and sperm functions. SEM study revealed the formation of deep wrinkles and folds on the testicular surface of the testes of beetles fed on treated diets, points towards the depletion of internal cellular material. The results suggest that egg white protein affects the survival and cause subsequent derangements in the testis of red flour beetle.

Proteomic identification of IPSE/alpha-1 as a major hepatotoxin secreted by Schistosoma mansoni eggs.

BACKGROUND: Eggs deposited in the liver of the mammalian host by the blood fluke parasite, Schistosoma mansoni, normally drive a T-helper-2 (Th2)-mediated granulomatous response in immune-competent mice. By contrast, in mice deprived of T-cells and incapable of producing granulomata, egg-secreted proteins (ESP) induce acute hepatic injury and death. Previous work has shown that one such ESP, the T2 ribonuclease known as omega-1, is hepatotoxic in vivo in that specific antisera to omega-1 prevent hepatocyte damage. METHODOLOGY/PRINCIPAL FINDINGS: Using an in vitro culture system employing mouse primary hepatocytes and alanine transaminase (ALT) activity as a marker of heptocyte injury, we demonstrated that S. mansoni eggs, egg-secreted proteins (ESP), soluble-egg antigen (SEA), and omega-1 are directly hepatotoxic and in a dose-dependent manner. Depletion of omega-1 using a monoclonal antibody abolished the toxicity of pure omega-1 and diminished the toxicity in ESP and SEA by 47 and 33%, respectively. Anion exchange chromatography of ESP yielded one predominant hepatotoxic fraction. Proteomics of that fraction identified the presence of IPSE/alpha-1 (IL-4 inducing principle from S. mansoni eggs), a known activator of basophils and inducer of Th2-type responses. Pure recombinant IPSE/alpha-1 also displayed a dose-dependent hepatotoxicity in vitro. Monoclonal antibody depletion of IPSE/alpha-1 abolished the latter's toxicity and diminished the total toxicity of ESP and SEA by 32 and 35%, respectively. Combined depletion of omega-1 and IPSE/alpha-1 diminished hepatotoxicity of ESP and SEA by 60 and 58% respectively. CONCLUSIONS: We identified IPSE/alpha-1 as a novel hepatotoxin and conclude that both IPSE/alpha-1 and omega-1 account for the majority of the hepatotoxicity secreted by S. mansoni eggs.

Preparation, characterization and evaluation of breviscapine lipid emulsions coated with monooleate-PEG-COOH.

Series of monooleate-modified PEG with active carboxylic terminus on the other end (MO-PEG-COOH) were used to modify the lipid emulsions surface to prepare a sterically stabilized lipid emulsions for carrying Traditional Chinese Medicine - breviscapine. Based on the research of relationship between polymer structure and prolonged circulation activity, we developed an optimized formulation and a technological method to prepare the sterile and stable MO-PEG(10,000)-COOH (Bre-LE-PEG(10,000)) coated breviscapine lipid emulsions (Bre-LE) for intravenous administration. Follow the optimum preparation, the average particle size, polydispersity index, zeta potential, Ke value and content of final product were determined to be (207.1±8.5)nm, 0.197±0.005, (-33.6±2.0)mV, (21.1±2.3)% and (95.0±1.8)% respectively (n=3). The characteristics, stability and safety of Bre-LE-PEG(10,000) were also studied with Bre-LE as a control. Increased plasma concentration by surface modification of the lipid emulsions may enhance the pharmacological activity of breviscapine to promote blood circulation.

First egg protein with a neurotoxic effect on mice.

While many invertebrates sequester toxic compounds to endow eggs with chemical defences, here we show, for the first time to our knowledge, the identification of a neurotoxin of proteinaceous nature localized inside an egg. Egg extracts from the freshwater apple snail Pomacea canaliculata displayed a neurotoxic effect in mice upon intraperitoneal injection (i.p.) (LD50, 96h 2.3mg/kg). Egg protein and total lipids were analysed separately and the only fraction displaying a highly toxic effect (LD50, 96h 0.25mg/kg, i.p.) was further purified to homogeneity as an oligomeric glyco-lipoprotein of 400kDa and two subunits biochemically and immunologically indistinguishable from the previously described perivitellin PV2. The neurotoxin was heat sensitive and there was evidence of circulating antibody response to sublethal i.p. doses on mice. Clinical signs, histopathological and immunocytochemical studies revealed damage mostly in mice spinal cord. Experiments showed chromatolysis and a decreased response to calbindin D-28K associated with a significant increase of TUNEL-positive cells in the dorsal horn neurons. These results suggest that calcium buffering and apoptosis may play a role in the neurological disorders induced by the toxin in mammalian central nervous system. This is the first report of a mollusc neurotoxin genetically encoded outside the cone-snail species.

Role for NMDA receptors in visceral nociceptive transmission in the anterior cingulate cortex of viscerally hypersensitive rats.

We have identified colorectal distension (CRD)-responsive neurons in the anterior cingulate cortex (ACC) and demonstrated that persistence of a heightened visceral afferent nociceptive input to the ACC induces ACC sensitization. In the present study, we confirmed that rostral ACC neurons of sensitized rats [induced by chicken egg albumin (EA)] exhibit enhanced spike responses to CRD. Simultaneous in vivo recording and reverse microdialysis of single ACC neurons showed that a low dose of glutamate (50 microM) did not change basal ACC neuronal firing in normal rats but increased ACC neuronal firing in EA rats from 18 +/- 2 to 32 +/- 3.8 impulses/10 s. A high dose of glutamate (500 microM) produced 1.95-fold and a 4.27-fold increases of ACC neuronal firing in sham-treated rats and in EA rats, respectively, suggesting enhanced glutamatergic transmission in the ACC neurons of EA rats. Reverse microdialysis of the 3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainite receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) reduced basal and abolished CRD-induced ACC neuronal firing in normal rats. In contrast, microdialysis of N-methyl-d-aspartate (NMDA) receptor antagonist AP5 had no effect on ACC neuronal firing in normal rats. However, AP5 produced 86% inhibition of ACC neuronal firing evoked by 50 mmHg CRD in the EA rats. In conclusion, ACC nociceptive transmissions are mediated by glutamate AMPA receptors in the control rats. ACC responses to CRD are enhanced in viscerally hypersensitive rats. The enhancement of excitatory glutamatergic transmission in the ACC appears to mediate this response. Furthermore, NMDA receptors mediate ACC synaptic responses after the induction of visceral hypersensitivity.

CTLA-4 ablation and interleukin-12 driven differentiation synergistically augment cardiac pathogenicity of cytotoxic T lymphocytes.

CD8+ cytotoxic T lymphocytes contribute to viral and autoimmune myocarditis and cardiac allograft rejection. The role of cytotoxic T-lymphocyte-associated antigen (CTLA)-4 as a negative regulator of CD4+ T cells is well defined, yet CTLA-4 regulation of CD8+ T cells is less clear. We studied CTLA-4 regulation of cytotoxic T lymphocytes in a transgenic model of CD8+ T-cell-mediated myocarditis. We generated CTLA-4(-/-) Rag 2(-/-) OT-1 mice, the CD8+ T cells of which express an ovalbumin (OVA) peptide-specific, class I major histocompatibility complex-restricted T-cell receptor. CTLA-4(-/-Tc12) OT-1 effectors, differentiated with interleukin-12 present, are hyperproliferative in vitro, compared with CTLA-4(+/+)Tc12 OT-1 controls. Transfer of low doses of CTLA-4(-/-Tc12) OT-1 cells to cMy-mOVA mice, which express OVA on cardiac myocytes, causes severe myocarditis, with 99% mortality, compared with no mortality after transfer of low doses of CTLA-4(+/+)Tc12 OT-1 cells. High doses of CTLA-4(+/+)Tc12 cells cause lethal myocarditis in cMy-mOVA mice, but high doses of CTLA-4(+/+)Tc0 CTL, generated without interleukin-12, are hypoproliferative within the cardiac-draining lymph node and do not significantly infiltrate the heart. In contrast, CTLA-4(-/-Tc0) cytotoxic T lymphocytes do proliferate in the cardiac-draining lymph node and diffusely infiltrate the heart. Nonetheless, high doses of CTLA-4(-/-Tc0) cells cause only limited tissue damage, and the disease is not lethal. These data show that CTLA-4 regulates myocarditic CD8+ T cell responses and that CTLA-4 deficiency partly overcomes a differentiation block that exists when naïve CD8+ T cells are stimulated without interleukin-12. Therefore, targeting CTLA-4 solely or in conjunction with interleukin-12 could influence effector CD8+ T cell responses in therapeutically beneficial ways.

Vitellogenin and zona radiata proteins as biomarkers of endocrine disruption in peregrine falcon (Falco peregrinus).

The aim of this study was to test a specific method for the detection of Vitellogenin (Vtg) and Zona Radiata Proteins (Zrp) in plasma from peregrine falcon (Falco peregrinus) as specific biomarkers for the evaluation of the effects of endocrine disruptors. The method was assayed with different peregrine falcon individuals (including mature and immature birds of both sexes) from a Spanish population being studied in terms of their contamination with organochlorine compounds with endocrine disrupting properties. This study shows that mouse anti bird Vtg monoclonal antibody ND3C3 (Biosense) seems to be the most specific antibody in binding plasmatic lipoproteins in peregrine falcon when compared to other anti Vtg antibodies. Rabbit anti salmon Zrp polyclonal antibodies O146 (Biosense) show cross-reactivity with Zrp in the samples studied. These preliminary results confirm the applicability of both of these diagnostic tools assayed (induction of Vtg and Zrp) in detecting exposure to Endocrine Disrupting Chemicals (EDCs) in this species. The increase of Vtg and Zrp detected in male specimens suggest a potential hazard to EDCs in the peregrine falcon which represents a species still affected by organochlorine compounds, and in particular those with estrogenic activity.

RNA damage and inhibition of neoplastic endothelial cell growth: effects of human and amphibian ribonucleases.

Angiogenesis defines the many steps involved in the growth and migration of endothelial cell-derived blood vessels. This process is necessary for the growth and metastasis of tumors, and considerable effort is being expended to find inhibitors of tumor angiogenesis. This usually involves screening of potential anti-angiogenic compounds on endothelial cells. To this end, two candidate anti-angiogenic RNA-damaging agents, onconase and (-4)rhEDN, were screened for their effects on endothelial cell proliferation using three distinct types of endothelial cells in culture: HPV-16 E6/E7-immortalized human umbilical vein endothelial cells (HUVECs), a Kras-transformed HPV-16 E6/E7 HUVEC (Rhim et al., Carcinogenesis 4, 673-681, 1998), and primary HUVECs. Onconase similarly inhibited proliferation in all three cell lines (IC(50) = 0.3-1.0 microM) while (-4)rhEDN was more effective on immortalized HUVEC cell lines (IC(50) = 0.02-0.06 microM) than on primary HUVECs (IC(50) > 0.1 microM). Differential sensitivity to these agents implies that more than one endothelial cell type must be used in proliferation assays to screen for novel anti-angiogenic compounds.

Enhanced cellular radiation sensitivity of androgen-independent human prostate tumor cells by onconase.

The RNase-like onconase, isolated from amphibian oocytes, showed increases in median tumor pO2 in solid tumors (1). This led us to consider if onconase could decrease cellular O2 consumption (QO2) on 9L rat glioma as well as DU145 human prostate adenocarcinoma cells. Using a Clark-type electrode chamber, we observed that onconase significantly inhibited QO2 in both tumors we tested. Since onconase-induced reduction in QO2 could lead to increases in radiation sensitivity, due to the diffusion of O2 to previously hypoxic tumor cells, we used androgen-insensitive DU145 cells to study onconase-induced changes in radiation sensitivity in vitro. Radiation sensitization was achieved with > 5 micrograms/ml of onconase, regardless of the p53 status of tumor cells. Data presented here suggested that onconase-induced enhancement in radiation sensitization in vitro of androgen-insensitive prostate cancer cells warranted further studies of radiation responses in vivo, prior to clinical settings for the advanced-stages of prostate cancer.

A ribonuclease A variant with low catalytic activity but high cytotoxicity.

Onconase, a homolog of ribonuclease A (RNase A) with low ribonucleolytic activity, is cytotoxic and has efficacy as a cancer chemotherapeutic. Here variants of RNase A were used to probe the interplay between ribonucleolytic activity and evasion of the cytosolic ribonuclease inhibitor protein (RI) in the cytotoxicity of ribonucleases. K41R/G88R RNase A is a less active catalyst than G88R RNase A but, surprisingly, is more cytotoxic. Like Onconase, the K41R/G88R variant has a low affinity for RI, which apparently compensates for its low ribonucleolytic activity. In contrast, K41A/G88R RNase A, which has the same affinity for RI as does the K41R/G88R variant, is not cytotoxic. The nontoxic K41A/G88R variant is a much less active catalyst than is the toxic K41R/G88R variant. These data indicate that maintaining sufficient ribonucleolytic activity in the presence of RI is a requirement for a homolog or variant of RNase A to be cytotoxic. This principle can guide the design of new chemotherapeutics based on homologs and variants of RNase A.

Conformational stability is a determinant of ribonuclease A cytotoxicity.

Onconasetrade mark, a homolog of bovine pancreatic ribonuclease A (RNase A) with high conformational stability, is cytotoxic and has efficacy as a cancer chemotherapeutic agent. Unlike wild-type RNase A, the G88R variant is toxic to cancer cells. Here, variants in which disulfide bonds were removed from or added to G88R RNase A were used to probe the relationship between conformational stability and cytotoxicity in a methodical manner. The conformational stability of the C40A/G88R/C95A and C65A/C72A/G88R variants is less than that of G88R RNase A. In contrast, a new disulfide bond that links the N and C termini (residues 4 and 118) increases the conformational stability of G88R RNase A and C65A/C72A/G88R RNase A. These changes have little effect on the ribonucleolytic activity of the enzyme or on its ability to evade the cytosolic ribonuclease inhibitor protein. The changes do, however, have a substantial effect on toxicity toward human erythroleukemia cells. Specifically, conformational stability correlates directly with cytotoxicity as well as with resistance to proteolysis. These data indicate that conformational stability is a key determinant of RNase A cytotoxicity and suggest that cytotoxicity relies on avoiding proteolysis. This finding suggests a means to produce new cancer chemotherapeutic agents based on mammalian ribonucleases.

Molecular determinants of apoptosis induced by the cytotoxic ribonuclease onconase: evidence for cytotoxic mechanisms different from inhibition of protein synthesis.

Cytotoxic endoribonucleases (RNases) possess a potential for use in cancer therapy. However, the molecular determinants of RNase-induced cell death are not well understood. In this work, we identify such determinants of the cytotoxicity induced by onconase, an amphibian cytotoxic RNase. Onconase displayed a remarkable specificity for tRNA in vivo, leaving rRNA and mRNA apparently undamaged. Onconase-treated cells displayed apoptosis-associated cell blebbing, nuclear pyknosis and fragmentation (karyorrhexis), DNA fragmentation, and activation of caspase-3-like activity. The cytotoxic action of onconase correlated with inhibition of protein synthesis; however, we present evidence for the existence of a mechanism of onconase-induced apoptosis that is independent of inhibition of protein synthesis. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone (zVADfmk), at concentrations that completely prevent apoptosis and caspase activation induced by ligation of the death receptor Fas, had only a partial protective effect on onconase-induced cell death. The proapoptotic activity of the p53 tumor suppressor protein and the Fas ligand/Fas/Fas-associating protein with death domain (FADD)/caspase-8 proapoptotic cascade were not required for onconase-induced apoptosis. Procaspases-9, -3, and -7 were processed in onconase-treated cells, suggesting the involvement of the mitochondrial apoptotic machinery in onconase-induced apoptosis. However, the onconase-induced activation of the caspase-9/caspase-3 cascade correlated with atypically little release of cytochrome c from mitochondria. In turn, the low levels of cytochrome c released from mitochondria correlated with a lack of detectable translocation of proapoptotic Bax from the cytosol onto mitochondria in response to onconase. This suggests the possibility of involvement of a different, potentially Bax- and cytochrome c-independent mechanism of caspase-9 activation in onconase-treated cells. As one possible mechanism, we demonstrate that procaspase-9 is released from mitochondria in onconase-treated cells. A detailed understanding of the molecular determinants of the cytotoxic action of onconase could provide means of positive or negative therapeutic modulation of the activity of this potent anticancer agent.

An oral sensitization model in Brown Norway rats to screen for potential allergenicity of food proteins.

We developed an oral sensitization protocol for food proteins for the rat. Young Brown Norway (BN) rats were exposed to 1 mg ovalbumin (OVA) by daily gavage dosing for 42 days without the use of an adjuvant. OVA-specific IgE and IgG responses were determined by ELISA. On an oral challenge with OVA some clinical symptoms of food allergy-like effects on the respiratory system, blood pressure, and permeability of the gastrointestinal barrier were studied. In addition, BN rats were orally exposed to a total hen egg white protein (HEW) extract and cow's milk (CM) and the specificities of induced antibody responses were compared with the specificities of antibodies in sera from egg- and milk-allergic patients using immunoblotting. Animals orally exposed to the allergens developed specific IgE and IgG antibodies which recognized the same proteins compared with antibodies from egg- or CM-allergic patients. Among the various clinical symptoms of food allergy, gut permeability was increased after an oral challenge. In addition, some animals demonstrated a temporary decrease in breathing frequency or systolic blood pressure. The results obtained show that the Brown Norway rat is a suitable animal model for inducing specific IgG and IgE responses on daily intragastric dosing of OVA without the use of an adjuvant. Moreover, local immune-mediated effects on oral challenge are observed. The observation that enterally exposed BN rats and food-allergic patients demonstrate antibody responses to a comparable selection of proteins on exposure to different protein mixtures (HEW and CM) further supports the suitability of the BN rat as an animal model for food allergy research and for the study of the allergenicity of (novel) food proteins.

Reversible nephrotoxicity of onconase and effect of lysine pH on renal onconase uptake.

PURPOSE: To examine the histopathology of the kidney in mice following repeated injections of the antitumor drug onconase, and to determine whether lysine, which reportedly blocks kidney uptake of other basic proteins, blocks the high renal uptake of onconase. METHODS: Mice received repeated intraperitoneal onconase injections over 3 weeks. Kidneys were examined by light microscopy after 1 week, 3 weeks, and 5 weeks (2 weeks after cessation of injections) and compared to kidneys from animals receiving a similar schedule of PBS injections. Renal uptake of radioiodinated onconase was measured in animals receiving intraperitoneal injections of lysine solutions of acidic and neutral pH given at -30, 0 and + 5 min relative to intravenous onconase injection. Renal onconase uptake was also measured in animals made metabolically acidotic by ingestion of ammonium chloride, arginine chloride or lysine dihydrochloride from the drinking water. RESULTS: Onconase caused acute moderate multifocal proximal renal tubular necrosis, and this toxicity was reversed by 2 weeks after drug withdrawal. Intraperitoneal injections of lysine dihydrochloride in PBS (pH 1.5) reduced renal onconase uptake at 15 min from 67.9+/-13.4% to 17.0+/-3.8% of the injected dose without affecting the plasma concentration and also reduced the fraction of degraded onconase in the urine. However, neutral solutions of lysine dihydrochloride at pH 7.4 or lysine acetate at pH 7.1 were ineffective at blocking renal onconase uptake. Furthermore, renal onconase uptake was minimally or not affected by a state of metabolic acidosis. CONCLUSIONS: Proximal tubular toxicity of onconase was reversible. Renal onconase uptake was blocked by lysine at pH 1.5 but not at neutral pH.

Cell cycle-related differences in susceptibility of NIH/3T3 cells to ribonucleases.

Microinjection of Onconase or RNase A into NIH/3T3 cells was used to study the intracellular actions of these two proteins. Onconase preferentially killed actively growing cells in both microinjection and cell culture experiments. Moreover, agents that increased the number of cells in S phase such as serum or microinjected signal transduction mediators (Ras, protein kinase C, and mitogen-activated protein kinase) enhanced Onconase cytotoxicity. Conversely, agents that decreased these proliferative pathways (dibutyryl cAMP and protein kinase A) correspondingly diminished Onconase cytotoxicity in microinjection experiments. These results were also mimicked in cell culture experiments since log-phase v-ras-transformed NIH/3T3 cells were more sensitive to Onconase (IC50 of 7 microg/ml) than parental NIH/3T3 fibroblasts (IC50 of 40 microg/ml). Based on those data we postulated that Onconase-mediated cell death in NIH/3T3 cells was related to events occurring at two or more points in the cell cycle preferentially associated with late G1/S and S phases. In contrast, quiescent NIH/3T3 cells were more sensitive to microinjected RNase A than log phase cells and positive mediators of proliferative signal transduction did not enhance RNase A-mediated cytotoxicity. Taken together, these results demonstrate that these two RNases use different pathways and/or mechanisms to elicit cytotoxic responses in NIH/3T3 cells. Predictions formulated from these studies can be tested for relevance to RNase actions in different target tumor cells.

Zona pellucida glycoprotein mZP3 produced in milk of transgenic mice is active as a sperm receptor, but can be lethal to newborns.

Mouse egg zona pellucida glycoprotein mZP3 (approximately 83 kDa M(r)) serves as a species-specific sperm receptor and acrosome reaction-inducer during fertilization in mice. These biological activities are dependent on certain mZP3 serine/threonine- (O-) linked oligosaccharides present at the combining-site for sperm. In an attempt to produce large amounts of biologically active mZP3, we generated several transgenic mouse lines carrying the full-length mZP3 gene fused to the beta-casein gene promoter and transcription termination sequence. We found that different transgenic mouse lines have different amounts of recombinant mZP3 (approximately 63 kDa M(r)) in milk of lactating females, from approximately 0.3 to 3.5 micrograms/microliter of milk. In all cases, purified milk-mZP3 is active as a sperm receptor and acrosome reaction-inducer in vitro. Unexpectedly, we also found that development of litters from these transgenic mice is related to the amount of mZP3 in the mother's milk. In the most extreme case, litters from the highest expressers fail to live beyond about day-7 post partum unless placed immediately after birth with surrogate wild-type mothers. Litters from lower expressers initially display a complex phenotype that includes effects on hair and body growth, but some of the mice survive and, in time, are restored to a wild-type phenotype. These results demonstrate that relatively large amounts of biologically active mZP3 can be produced in transgenic mouse milk for structural and other studies, but that the presence of mZP3 in milk has dramatic developmental effects on litters, ranging from retarded hair and body growth to death of newborn pups.

Enhanced in vitro cytotoxicity and cytostasis of the combination of onconase with a proteasome inhibitor.

In proliferating cells the turnover rate of proteins responsible for regulation of the cell cycle progression, namely cyclins and inhibitors of the cyclin-dependent kinases (CDKs) and phosphatases, is rapid and their cellular level is modulated at the transcriptional, translational and/or degradation (via proteasome pathway) stages. Inhibition of proteasome function results in accumulation of rapidly turning over proteins and, thus, causes an imbalance of the cell cycle regulatory components, and loss of their regulatory function. Indeed, it has been shown that proteasome inhibitors perturb the cell cycle progression. Onconase, a novel RNase which has anti-tumor activity and is in clinical trials, has previously been shown to suppress protein synthesis, presumably by degradation of intracellular RNA, preferentially tRNA. By interfering with regulation of expression of cyclins and/or CDK-inhibitors, onconase also may induce the imbalance of these proteins and potentiate the effect of proteasome inhibitors. In the present study, we observed that the combinations of onconase with peptide-aldehyde inhibitors of calpain and proteasome such as the N-acetyl-leucinyl-leucinyl-norleucinal (LLnL) and the N-acetyl-leucinyl-valinyl-phenylalaninal (LVP), but not N-acetyl-leucinyl-leucinyl-methioninal (LLM), were synergistic in suppressing cell proliferation and inducing apoptosis in three human tumor cell lines: A-549 lung adenocarcinoma, DU-145 prostatic carcinoma, and MDA-MB-231 breast carcinoma. The observed cytotoxicity may also be a result of prevention of the induction of the 'survival' genes by the nuclear factor kappaB (NFkappaB) by onconase and proteasome inhibitors. The data indicate that such combinations should be further tested as potential anti-cancer regimens.

The Rana catesbeiana rcr gene encoding a cytotoxic ribonuclease. Tissue distribution, cloning, purification, cytotoxicity, and active residues for RNase activity.

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found in R. catesbeiana (bullfrog) oocytes. It possesses both ribonuclease activity and cytotoxicity against tumor cells. We report here for the first time the cloning of RC-RNase cDNA from liver rather than from oocytes where RC-RNase is stored. An internal fragment of cDNA was obtained by reverse transcription-PCR using deduced oligonucleotides as primers. Full-length cDNA was obtained by 5'- and 3'-RACE technique. The cDNA clone, named rcr gene, contained a 5'-untranslated region, a putative signal peptide (22 amino acids), a mature protein (111 amino acids), a 3'-untranslated region, and a polyadenylation site. The cDNA which encoded the mature protein was fused upstream with a modified pelB signal peptide DNA and inserted into pET11d for expression in Escherichia coli strain BL21(DE3). The secretory RC-RNase in the culture medium was enzymatically active and was purified to homogeneity. The recombinant RC-RNase had the same amino acid sequence, specific activity, substrate specificity, antigenicity, and cytotoxicity as that of native RC-RNase from frog oocytes. Amino acid residues His-10, Lys-35, and His-103 are involved in RC-RNase catalytic activity. Ribonucleolytic activity was involved in and may be essential for RC-RNase cytotoxicity. DNA sequence analysis showed that RC-RNase had approximately 45% identity to that of RNase superfamily genes. This indicates that RC-RNase is a distinct ribonuclease gene in the RNase superfamily.