Pipiens Eggs Research Articles

Insecticidal effects of a novel polyherbal formulation (HF7) against Culex pipiens L. (Diptera: Culicidae).

Plant secondary metabolites represent the most efficient and convenient method to control and overcome environmental pollution and insecticidal resistance. This study explored the mosquitocidal activity of the combined extract of seven plants, (HF7) extracted using a Soxhlet extractor against Culex pipiens under laboratory conditions. Exposure of the 3rd instars of Cx. pipiens to HF7 hexane extract resulted in LC50:114.5 μg/mL and LC90:117.0 μg/mL values after 24 h. The ovicidal activities of hexane extract against Cx. pipiens eggs were 21.6%, 48.3%, and 71.6% at 187.5, 93.7, and 46.88 μg/mL, respectively. HF7-treated larvae showed the formation of irregular blebbing of epithelial cells toward the lumen and sloughing into the gut lumen. HF7 extract resulted in 100% adulticidal mortality at the concentration of 3.7 mg/test tube after 30 min of exposure. The IC50 of HF7 extract was 97.03 µg/ml against larvae, at which nuclear and morphological changes were observed. The spectroscopy spectrum of HF7 hexane extract disclosed the presence of 57 different secondary metabolites, among which the dominant compound was eugenol (32.3%). HF7 hexane extract could serve as a botanical insecticide for controlling Cx. pipiens and potentially other mosquito species.

Culex pipiens and Culex restuans egg rafts harbor diverse bacterial communities compared to their midgut tissues

BackgroundThe bacterial communities associated with mosquito eggs are an essential component of the mosquito microbiota, yet there are few studies characterizing and comparing the microbiota of mosquito eggs to other host tissues.MethodsWe sampled gravid female Culex pipiens L. and Culex restuans Theobald from the field, allowed them to oviposit in the laboratory, and characterized the bacterial communities associated with their egg rafts and midguts for comparison through MiSeq sequencing of the 16S rRNA gene.ResultsBacterial richness was higher in egg rafts than in midguts for both species, and higher in Cx pipiens than Cx. restuans. The midgut samples of Cx. pipiens and Cx. restuans were dominated by Providencia. Culex pipiens and Cx. restuans egg rafts samples were dominated by Ralstonia and Novosphingobium, respectively. NMDS ordination based on Bray-Curtis distance matrix revealed that egg-raft samples, or midgut tissues harbored similar bacterial communities regardless of the mosquito species. Within each mosquito species, there was a distinct clustering of bacterial communities between egg raft and midgut tissues.ConclusionThese findings expand the list of described bacterial communities associated with Cx. pipiens and Cx. restuans and the additional characterization of the egg raft bacterial communities facilitates comparative analysis of mosquito host tissues, providing a basis for future studies seeking to understand any functional role of the bacterial communities in mosquito biology.

Bioassay-guided fractionation of a dried commercial source, Alpinia galanga (L.) Willd rhizomes extract, against Culex pipiens (Diptera: Culicidae)

The increasing risk of insecticide resistance in mosquito populations has led to the search for new larvicidal agents. Evaluation of bioassay-guided fractionation of the rhizome extract of Alpinia galanga (L.) Willd against Aedes aegypti was assessed. Bioactive fractions were isolated from the rhizome extract of A. galanga using a Soxhlet extractor and chromatography techniques, and subsequently tested against the fourth instar of Culex pipiens. The lethal concentration (LC) was calculated via log-probit analysis. The active fraction was evaluated by gas chromatography-mass spectroscopy (GC–MS) and infrared (IR) analysis. The highest larvicidal potential obtained from bioassays using the Soxhlet apparatus was observed in dichloromethane (DCM) and ethyl acetate (EtoAc) extracts, with LC50 values of 124.49 and 176.30 ppm, respectively, after 24 h of exposure. Subsequently, the DCM extract was subjected to column and thin-layer chromatography. Results of the DCM extraction and the active TLC fraction (F133) of the Rf value 0.5 revealed that LC50 and LC90 values decreased over time. The F133 fraction of A. galanga exhibited zero hatchability (100% mortality) at LC50 (63.416 ppm) and LC25 (31.70 ppm) against Cx. pipiens eggs. GC–MS analysis of the active thin-layer chromatography TLC fraction (F133) revealed the presence of phenol 2 4-bis (1 1-dimethylethyl), which was identified as the major compound. Alpinia galanga extract is a promising candidate for the control of mosquito populations. Further study is required to determine the effect of the extracts on non-target organisms.

Efficacy of Two Monoterpenoids, Carvacrol and Thymol, and Their Combinations against Eggs and Larvae of the West Nile Vector Culex pipiens.

Background: Insect vector control is facing the challenges of resistance development and environmental hazards caused by synthetic pesticides. This has led to a considerable market opportunity for botanical insecticides. In this scenario, our study investigated the potential of selected bioactive monoterpenoids, carvacrol and thymol, as safe and effective tools to control the West Nile vector Culex pipiens. Furthermore, the combined effect of thymol-carvacrol mixtures and their possible interactions were assessed. Methods: For determining larvicidal and ovicidal 50% lethal concentration (LC50), each monoterpenoid was tested at different concentrations (5–500 mg/L). Then, the fixed ratio method was used for evaluating their combinational efficacy. Results: Carvacrol was more toxic against larvae of Cx. pipiens, with a LC50 value of 14 mg/L, whereas thymol exhibited a LC50 value of 49 mg/L. Comparable trends of efficacy were observed when toxicity on Cx. pipiens eggs was investigated, with LC50 values of 7 and 13 mg/L for carvacrol and thymol, respectively. In combinational toxicity assays, the mixture thymol-carvacrol at 1:4 ratio achieved a synergistic effect against larvae of Cx. pipiens, whereas an additive effect was observed on eggs. Other ratios showed antagonistic effects. Conclusions: Overall, our findings pointed out that the 1:4 ratio of thymol-carvacrol blend can enhance the insecticidal efficacy on Cx. pipiens young instars and can be considered further as active ingredient for developing botanical insecticides to be used in mosquito control operations.

Insecticidal Effect of Solenostemma argel Extracts Against Culex pipiens.

Of the various plant extracts from 10 plant species tested against larvae of Culex pipiens in the laboratory, only extracts from Solenostemma argel exhibited larvicidal activity. A chloroform leaf extract of S. argel exhibited relatively high activity with a lethal concentration causing 50% mortality (LC50) of 15.89 ppm, while chloroform and ethyl acetate extracts of S. argel fruits were 19.70 and 19.52 ppm, respectively. The chloroform fruit extract at 10 ppm reduced the hatchability of Cx. pipiens eggs by 20%, whereas the chloroform leaf extract was found to be less effective (5% reduction). At 10 ppm, adult emergence was reduced by 84% and 75% for chloroform and ethyl acetate extracts of fruits, respectively. Metamorphosis of larvae exposed to chloroform fruit extract (10 ppm) was extended to 15 days, as compared to 10 days for control larvae. It took 12 days at 1 ppm, and 15 days at 6 and 10 ppm for chloroform fruit extract-treated embryos to develop into adult mosquito while it took 10 days in the control treatment. However, 100% toxicity was observed in the embryos of zebrafish, Danio rerio, treated with the ethyl acetate fruit extracts (LC50 of 20 ppm and LC100 of 40 ppm) and chloroform leaf extract (LC50 of 30 ppm and LC100 of 60 ppm). These findings emphasize the need to further isolate the bioactive molecules in S. argel crude extracts that may still be mosquitocidal but produce no, or minimal, adverse effects on nontarget organisms such as zebrafish.

West Nile Virus Positive Blood Donation and Subsequent Entomological Investigation, Austria, 2014

The detection of West Nile virus (WNV) nucleic acid in a blood donation from Vienna, Austria, as well as in Culex pipiens pupae and egg rafts, sampled close to the donor’s residence, is reported. Complete genomic sequences of the human- and mosquito-derived viruses were established, genetically compared and phylogenetically analyzed. The viruses were not identical, but closely related to each other and to recent Czech and Italian isolates, indicating co-circulation of related WNV strains within a confined geographic area. The detection of WNV in a blood donation originating from an area with low WNV prevalence in humans (only three serologically diagnosed cases between 2008 and 2014) is surprising and emphasizes the importance of WNV nucleic acid testing of blood donations even in such areas, along with active mosquito surveillance programs.

Ovicidal activity of three insect growth regulators against Aedes and Culex mosquitoes

Interspecific variations in the susceptibility of freshly and embryonated eggs of Aedes albopictus, Ae. aegypti, Ae. atropalpus and Culex pipiens were tested against three classes of insect growth regulators (IGRs) including ecdysone agonist (azadirachtin), chitin synthesis inhibitor (diflubenzuron) and juvenile hormone analog (pyriproxyfen) at 0.001, 0.01, 0.1 and 1.0ppm concentrations. Egg hatching inhibition was dose dependent, the highest being at 1.0ppm concentration for freshly laid eggs of Ae. albopictus (pyriproxyfen: 80.6%, azadirachtin: 42.9% and diflubenzuron: 35.8%). Aedes aegypti showed lower egg hatching inhibition when exposed to pyriproxyfen (47.3%), azadirachtin (15.7%) and diflubenzuron (25.5%). Freshly laid eggs of Cx. pipiens were most susceptible to diflubenzuron. Aedes atropalpus eggs were tolerant to all three classes of IGRs. Embryonated eggs of Ae. albopictus, Ae. aegypti, Ae. atropalpus and Cx. pipiens were resistant to pyriproxyfen, azadirachtin and diflubenzuron than freshly laid eggs. The median desiccation time (DT50) of Ae. atropalpus eggs was maximum (5.1h) as compared to Ae. aegypti (4.9h), Ae. albopictus (3.9h) or Cx. pipiens (1.7h) eggs. Insignificant relationship between the rates of desiccation and egg hatching inhibition suggests other factors than physical providing eggs the ability to tolerate exposures to various IGRs. Egg hatching inhibition was due to the alteration in embryonic development caused by IGRs. Changes in the egg shell morphology and abnormal egg hatching from the side of the egg wall instead of operculum, was observed at higher concentrations of diflubenzuron. Morphological and physiological variations in eggs may be the key factor to influence the ovicidal efficacy of IGRs. The present data provide a base line for the improvement of the ovicidal efficacy of the insecticide and its formulation.

Insect-Specific Flaviviruses from Culex Mosquitoes in Colorado, with Evidence of Vertical Transmission

Mosquitoes were collected in Colorado during 2006 and 2007 to examine spatial and seasonal patterns of risk for exposure to Culex vectors and West Nile virus. We used universal flavivirus primers to test pools of Culex mosquitoes for viral RNA. This led to the detection and subsequent isolation of two insect-specific flaviviruses: Culex flavivirus (CxFV), which was first described from Japan, and a novel insect flavivirus, designated Calbertado virus (CLBOV), which has also been detected in California and Canada. We recorded both viruses in Cx. tarsalis and Cx. pipiens from Colorado. Furthermore, quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed the presence of CxFV RNA in Cx. pipiens eggs and larvae from a laboratory colony established in 2005 and naturally infected with CxFV, suggesting vertical transmission as a means of viral maintenance in natural Culex populations. Finally, we present phylogenetic analyses of the relationships between insect-specific flaviviruses and other selected flaviviruses.

Gonadotropin stimulation of steroid synthesis and metabolism in the Rana pipiens ovarian follicle: Sequential changes in endogenous steroids during ovulation, fertilization and cleavage stages

Steroid synthesis and metabolism have been followed in Rana pipiens ovarian follicles, denuded oocytes and eggs during ovulation, fertilization and cleavage stages (blastula formation). Under physiological conditions, gonadotropin stimulation of the fully grown follicle leads to progesterone synthesis from [ 3H]acetate as well as formation of much smaller amounts of 17α-hydroxyprogesterone, androstenedione, pregnanedione and pregnanediol. Progesterone levels increase during completion of the first meiotic division, but by ovulation progesterone disappears from the egg. Plasma membrane-bound progesterone is taken up into the oocyte cortical granules and is largely metabolized to 5α-pregnane-3αol,20-one and 5β-pregnane-3α,17α,20β-triol coincident with internalization of 60% of the oocyte surface (and >90% of bound progesterone) by the end of the hormone-dependent period. The principal steroid in the ovulated egg is 5β-pregnane-3α,17α,20β-triol. There is a rapid efflux of 5β-pregnane-3α,17α,20β-triol into the medium immediately following fertilization and residual steroid levels remain low in the developing blastula. Dissociated blastulae cells prepared from stage 9 1/2 embryos concentrate both pregnenolone and progesterone from the medium with minimal metabolism. The results indicate that the ovarian follicle has the ability to synthesize and metabolize progesterone but that this ability disappears in the ovulated egg. The progesterone metabolites formed during meiosis are largely released at fertilization.

OVIPOSITION PREFERENCES OF CULEX RESTUANS AND CULEX PIPIENS (DIPTERA: CULICIDAE) FOR SELECTED INFUSIONS IN OVIPOSITION TRAPS AND GRAVID TRAPS

Field studies were conducted in southwestern Virginia to determine the ovipositional preferences of Culex restuans and Culex pipiens by using ovitraps and gravid traps baited with selected infusions. For the ovitrap collections, 4 different infusions (manure, hay, grass, and rabbit chow) were used. Significant differences among infusions were detected on most sample dates for both species. For 3 of the first 4 wk of collections, the manure infusion collected significantly more Cx. restuans than all the other infusions. The hay and grass infusions collected the majority of the egg rafts during weeks 5-9. Cx. pipiens egg rafts were absent from the first 3 wk of collections. Of the remaining 6 wk, 4 showed significant differences in attractiveness of infusions, with the hay and grass infusions preferred by Cx. pipiens. Two infusions, manure and hay, were used for the gravid trap experiment and both Cx. restuans and Cx. pipiens data were combined for analysis. Only the first 2 wk showed significance, with manure being preferred over hay in both weeks. In later collections, the relative attractiveness of the hay infusion increased. A seasonal shift in infusion preference may be related to incubation temperature during preparation of the infusions. New infusions were prepared each week and incubation was done outside. Increased attractiveness of the hay infusion coincided with higher average temperatures in July and August. Hay infusion was very effective for trapping both Cx. pipiens and Cx. restuans in southwestern Virginia and is more convenient to use than manure. However, cool outside temperatures in the early season may interfere with the fermentation process and thus incubation should be done for a longer time or brought indoors.

Contamination and Wildlife Communities in Stormwater Detention Ponds in Guelph and the Greater Toronto Area, Ontario, 1997 and 1998 Part II — Contamination and Biological Effects of Contamination

Abstract Due to concerns that wildlife may be attracted to Stormwater detention ponds and would be exposed to contaminants accumulating in these ponds, a field study of 15 Stormwater ponds and one natural wetland in Guelph and the Greater Toronto Area, Ontario, was performed in 1997 and 1998. At 9 of 16 sites, copper levels in water exceeded the Canadian Water Quality Guidelines (CWQGs) for the protection of aquatic life. Canadian Water Quality Guidelines were also exceeded by average water concentrations for lead at four sites, for zinc at three sites, for chromium at seven sites, and turbidity at ten sites. At the natural wetland, only turbidity levels exceeded the CWQGs. Sediments from all ponds contained concentrations of at least one contaminant that exceeded the “lowest effects level” (LED of the Guidelines for the Protection and Management of Aquatic Sediment in Ontario. Stormwater pond sediments contained concentrations exceeding provincial sediment quality guidelines at the LEL for polychlorinated biphenyls (PCBs), gamma-hexachlorocyclohexane (g-bhc), total Kjeldahl nitrogen (TKN) and total phosphorus (TP), turbidity, oil and grease, total PAHs, chromium, zinc, cadmium, copper, lead, nickel and arsenic. Concentrations of g-bhc, TKN, TP, cadmium, copper, lead, zinc and arsenic in the sediment of the natural wetland also exceeded the LEL. Sediments at one site exceeded the provincial guideline at the severe effect level for chromium. Concentrations of lead, zinc, cadmium and total PAHs in sediments were correlated with the age of the ponds. Concentrations of organochlorine pesticides and PCBs were low (<0.08 µg/g wet weight) in pump-kinseed (Lepomis gibbosus) collected from two ponds in Guelph. Red-winged blackbird [Agelaius phoeniceus) eggs from two ponds in Toronto contained up to 1130 ng/g pp’DDE and up to 670 ng/g total PCBs. Those concentrations were much higher than in a reference site, but an order of magnitude below concentrations associated with health effects in songbirds. Among four Stormwater ponds and one natural wetland in Guelph, there was statistically lower hatching success of northern leopard frogs (Rana pipiens) eggs, and larvae took longer to metamorphose into frogs at two Stormwater ponds. Statistically fewer tadpoles survived to metamorphosis at one Stormwater pond. We conclude that Stormwater ponds do not offer clean ecosystems for wildlife and the monitoring of contamination and its effects within Stormwater ponds is necessary.

Anuran Development, Density and Diversity in Relation to Agricultural Activity in the Holland River Watershed, Ontario, Canada (1990–1992)

Significantly lower anuran species diversity and density was recorded in a vegetable growing area relative to upstream and downstream sites in the Holland River watershed, Ontario, Canada. Egg hatching success and tadpole deformity rates of American toads (Bufo americanus americanus), green frogs (Rana clamitans melanota) and northern leopard frogs (Rana pipiens) eggs in water from field sites and control water were assessed. Compared to the control and the upstream site, the total abnormality rate (unhatched eggs plus deformed tadpoles) was higher for American toads in water from the agricultural and downstream sites. Total abnormality was higher in green frog eggs in water from the agricultural site and a downstream site. Trace concentrations of organophosphorus pesticides were detected most often in agricultural zone water and sediments than in upstream and downstream sites. Organochlorine pesticide residues, especially in agricultural zone samples, exceeded the no effect level guidelines for the protection of aquatic life. Ammonia, phosphorus, particulates, BOD and TKN were highest in the agricultural zone. Significant correlations between these parameters and anuran development suggest nutrient run-off as a causal or contributing factor in lower anuran diversity, density and reproductive success of American toads and green frogs in the site dominated by agriculture.

Role of amphibian egg transglutaminase in the development of secondary cytostatic factor in vitro

Fresh cytosols extracted from unfertilized amphibian eggs contain a cytostatic factor (CSF) which arrests the cell cycle at metaphase when microinjected into cleaving blastomeres. This CSF is sensitive to Ca2+, and is designated primary CSF (1°CSF). During storage of Ca2+-containing cytosols at 2°C, stable CSF activity appears, designated secondary CSF (2°CSF). In Rana pipiens egg cytosols, the development of 2°CSF coincides with the formation of a protein complex with a molecular weight above 2,000 kDa, and this large molecule exhibits a high 2°CSF activity when purified (Shibuya and Masui, 1989: Development 106:799–808). The present study shows that both the formation of 2°CSF protein complex and the development of its activity are inhibited by ethylamine and glycine-ethyl-ester (GEE), both known as potent transglutaminase (TGase) inhibitors. An affinity-purified polyclonal antibody raised against mammalian transglutaminase reacts with an approximately 68-kDa protein in fresh egg cytosols, as well as with the 2°CSF protein complex. In cytosols deprived of transglutaminase by immunoprecipitation, neither the development of 2°CSF activity nor the formation of its protein complex can occur. These results indicate that transglutaminase of Rana pipiens eggs is responsible for the formation of 2°CSF, and that transglutaminase itself is incorporated into 2°CSF molecules. Mol. Reprod. Dev. 47:302–311, 1997. © 1997 Wiley-Liss, Inc.

Role of amphibian egg transglutaminase in the development of secondary cytostatic factor in vitro.

Fresh cytosols extracted from unfertilized amphibian eggs contain a cytostatic factor (CSF) which arrests the cell cycle at metaphase when microinjected into cleaving blastomeres. This CSF is sensitive to Ca2+, and is designated primary CSF (1 degree CSF). During storage of Ca(2+)-containing cytosols at 2 degrees C, stable CSF activity appears, designated secondary CSF (2 degrees CSF). In Rana pipiens egg cytosols, the development of 2 degrees CSF coincides with the formation of a protein complex with a molecular weight above 2,000 kDa, and this large molecule exhibits a high 2 degrees CSF activity when purified (Shibuya and Masui, 1989: Development 106:799-808). The present study shows that both the formation of 2 degrees CSF protein complex and the development of its activity are inhibited by ethylamine and glycine-ethyl-ester (GEE), both known as potent transglutaminase (TGase) inhibitors. An affinity-purified polyclonal antibody raised against mammalian transglutaminase reacts with an approximately 68-kDa protein in fresh egg cytosols, as well as with the 2 degrees CSF protein complex. In cytosols deprived of transglutaminase by immunoprecipitation, neither the development of 2 degrees CSF activity nor the formation of its protein complex can occur. These results indicate that transglutaminase of Rana pipiens eggs is responsible or the formation of 2 degrees CSF, and that transglutaminase itself is incorporated into 2 degrees CSF molecules.

Local alteration of cortical actin in Xenopus eggs by the fertilizing sperm

Rhodamine phalloidin (Rph) staining was used to examine the microfilament organization of the Xenopus laevis egg cortex during the early stages of fertilization. Unactivated eggs possessed a cytochalasin B (CB)-insensitive Rph-stained matrix that was reorganized upon egg activation and diminished in the presence of CB. Xenopus laevis sperm caused a temporary local increase in Rph staining on the Xenopus cortex. In CB-treated eggs, the local increases of cortical Rph staining later changed to a Rph-free area. These temporary local increases of cortical Rph staining were also observed when Notophthalmus viridescens sperm fertilized Xenopus and Rana pipiens eggs, and were followed by the appearance of concentric rings of stained and unstained areas. Our data suggest that Xenopus and Notophthalmus sperm have activities that can both organize and disrupt the cortical filamentous actin of the Xenopus egg.

Cytotoxic onconase and ribonuclease a chimeras: comparison andin vitrocharacterization

AbstractOnconase, a protein isolated from fertilized Rana pipiens eggs, has antineoplastic properties both in vitro (Z. Darzynkiewicz, S. P. Carter, S. M. Mikulski, W. Ardelt, and K. Shogen, 1988, Cell Tissue Kinet. 21, 169–182) and in vivo (S. M. Mikulski, W. Ardelt, K. Shogen, E. H. Bernstein, and H. Menduke, 1990, J. Natl. Cancer Inst. 82, 151–153). The protein is a member of the pancreatic ribonuclease A superfamily and exhibits ribonuclease activity toward highly polymerized RNA (W. Ardelt, S. M. Mikulski, and K. Shogen, 1991, J. Biol. Chem. 266, 245–251). Previous work has demonstrated that bovine pancreatic ribonuclease A conjugated to transferrin (S. Rybak, S. Saxena, E. Ackerman, and R. Youle, 1991, J. Biol. Chem. 266, 21202–21207) or antibodies to the human transferrin receptor (D. Newton, O. Ilercil, D. Laske, E. Oldfield, S. Rybak, and R. Youle, 1992, J. Biol. Chem. 267, 169–182), acted like a class of reagents called immunotoxins to specifically inhibit protein synthesis in ligand-positive ce...

Evidence for the involvement of microtubules, ER, and kinesin in the cortical rotation of fertilized frog eggs.

During the first cell cycle, the vegetal cortex of the fertilized frog egg is translocated over the cytoplasm. This process of cortical rotation creates regional cytoplasmic differences important in later development, and appears to involve an array of aligned microtubules that forms transiently beneath the vegetal cortex. We have investigated how these microtubules might be involved in generating movement by analyzing isolated cortices and sections of Xenopus laevis and Rana pipiens eggs. First, the polarity of the cortical microtubules was determined using the "hook" assay. Almost all microtubules had their plus ends pointing in the direction of cortical rotation. Secondly, the association of microtubules with other cytoplasmic elements was examined. Immunofluorescence revealed that cytokeratin filaments coalign with the microtubules. The timing of their appearance and their position on the cytoplasmic side of the microtubules suggested that they are not involved directly in generating movement. ER was visualized with the dye DiIC16(3) and by immunofluorescence with anti-BiP (Bole, D. G., L. M. Hendershot, and J. F. Kearney, 1986. J. Cell Biol. 102:1558-1566). One layer of ER was found closely underlying the plasma membrane at all times. An additional, deeper layer formed in association with the microtubules of the array. Antibodies to sea urchin kinesin (Ingold, A. L., S. A. Cohn, and J. M. Scholey. 1988. J. Cell Biol. 107:2657-2667) detected antigens associated with both the ER and microtubules. On immunoblots they recognized microtubule associated polypeptide(s) of approximately 115 kD from Xenopus eggs. These observations are consistent with a role for kinesin in creating movement between the microtubules and ER, which leads in turn to the cortical rotation.

Tamoxifen and trifluoroperazine (Stelazine) potentiate cytostatic/cytotoxic effects of P‐30 protein, a novel protein possessing anti‐tumour activity

P-30 protein, a novel protein isolated in our laboratory from fertilized Rana pipiens eggs, has been shown to possess significant anti-proliferative and cytotoxic activity against a variety of human tumour cell lines. This protein also shows a potent anti-tumour activity in vivo in animal tumour models and is currently undergoing Phase I human clinical trials in cancer patient volunteers. The present study describes the in vitro effects of the concerted action of this protein and two other agents which affect the cell proliferative cycle. A significant potentiation of the P-30 protein-induced cell growth inhibition by tamoxifen as well as trifluoroperazine (Stelazine) in both the human A-549 lung carcinoma and the ASPC-1 pancreatic adenocarcinoma systems at wide ranges of drug concentrations was observed. The effect was apparently due to the synergistic action of P-30 protein and the agents tested. This data may provide clues that can be useful in explaining the mechanism of its anti-tumour activity. The results are also helpful for the designing in vivo animal and, perhaps eventually, human studies, whereby the combination therapies utilizing P-30 protein with agents of relatively low toxicity such as tamoxifen and/or Stelazine could offer a promising treatment(s) for these notoriously refractory types of human cancer.

Cytostatic factor (CSF) activity in cytosols extracted from Xenopus laevis eggs

Cytostatic factor (CSF), found in the cytoplasm of unfertilized eggs of amphibians, causes metaphase arrest when microinjected into cleaving blastomeres. Although CSF from Rana pipiens eggs has been extracted and characterized, little is known about CSF extracted from eggs of other species. We investigated the conditions required to preserve CSF activity in cytosols extracted from Xenopus laevis eggs and found that it was necessary to expose the eggs to CO2 prior to extraction and that the extraction buffer must contain sodium beta-glycerophosphate. CSF activity disappeared after 24 h of storage at 2 degrees C. Cytological examination showed that the arrested blastomeres injected with cytosols had been arrested at metaphase and contained a spindle lacking polar asters, in which highly condensed chromosomes were embedded.

Cell adhesion to extracellular matrix in normal Rana pipiens gastrulae and in arrested hybrid gastrulae Rana pipiens ♀ × Rana esculenta ♂

Rana pipiens eggs fertilized by Rana esculenta sperm (ESC) hybrid embryos develop until gastrulation in control Rana pipiens embryos (PIP) and then show morphogenetic arrest. After arrest, ESC do not gastrulate but live for 5 days as blastula-like embryos. We studied the distribution of fibronectin (FN)-containing fibrils and integrin (INT) in PIP and ESC. There are many FN-fibrils in PIP organized in anastomosing networks radiating away from the center of individual cells and across intercellular boundaries. ESC have fewer fibrils compared to PIP. These fibrils are first located between cells in disorganized arrays. After arrest in ESC, when PIP are Stage 14 neurulae, many more FN-fibrils appear. INT-staining occurs in both embryos in similar patterns. In xenoplastic transplantations, we found that the extracellular matrix on the inner surface of the ESC blastocoel roof serves as a substratum for PIP cell migration. In an in vitro assay, we found more cell adhesion to FN-substrata in PIP than in ESC. Cell locomotion rates on FN-substrata were 1.70 ± 0.85 μm/min for PIP but only 0.46 ± 0.56 μm/min for ESC. We also found that the inner surface of the blastocoel roof from ESC can not promote cell adhesion and locomotion when Stage 11 fragments are used for conditioning but that Stage 14 fragments can deposit a FN-fibril-rich extracellular matrix which supports PIP mesodermal cell migration at a rate of 1.26 ± 0.38 μm/min.