Aggregation and ion transfer induced by tentoxin

It is shown that tentoxin, a cyclic tetrapeptide with two N-methylated residues, is able, when added to lipid bilayers, to increase the transmembrane current through discrete events. Conformational investigations involving 1H-NMR, infrared and circular dichroism studies show that, at concentrations above 7 X 10(-5) M, the cyclic tetrapeptide aggregates in chloroform. We suggest that the aggregates could form a pore through a stacking of cycles.     

Cloned ferrets produced by somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (approximately 3-4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink.     

Sensitization of rat gastrointestinal tract to acetylcholine and histamine produced by X-radiation

Abdominal x-radiation produces both acute and chronic disturbances of gastrointestinal motility. Anaesthetized Albino-Oxford rats received one-session x-radiation (absorbed dose 10 Gy) of whole abdomen. Two hours after irradiation the rats were sacrificed and segments of their gastrointestinal tract (gastric fundus, jejunum, ileum and ascending colon, were mounted in isolated organ bath. Acetylcholine and 5-hydroxytryptamine produced tonic contractions of all gut segments, while histamine did so only with gastric fundus. While contractile effect of 5-hydroxytryptamine was not affected by x-radiation, the responses of all gut segments on acetylcholine were potentiated and shifted towards lower concentrations. After x-radiation histamine produced concentration-dependent tonic contraction of previously unresponsive jejunum and ascending colon. The results of our study suggest that x-radiation produces acute sensitization of rat gastrointestinal tract to acetylcholine and histamine.     

Blow fly responses to semiochemicals produced by decaying carcasses

Volatiles from mouse carcasses in decay stages ranging from fresh to 33 days old were used to investigate oriented flight and landings in male and female blow flies of Calliphora vicina Robineau‐Desvoidy (Diptera: Calliphoridae). Oriented flight increased significantly from 36% towards fresh carcasses to 68%, 61% and 65% towards carcasses aged 3 days, 6 days and 9 days, respectively. Carcasses aged 20 days and 33 days were significantly less attractive, achieving 51% and 41% attraction, respectively. No differences emerged between the sexes in oriented flight, but a significant increase in female landings at the most attractive carcasses was observed. Headspace collections from the different stages of decay showed a succession in the volatile profile emitted from the carcasses and identified nine chemicals which peak in quantity in concurrence with the most attractive stages of decay. Three of these chemicals also showed dose–response effects as indicated by a significant correlation between the amount present and the proportion of flies responding. Blow flies are important pests and efficient traps are needed. The significant interaction between fly sex and carcass age highlights behavioural differences between male and female blow flies which can be exploited in blow fly trapping. Three new volatile chemicals, butylated hydroxyl toluene, 3‐hydroxy‐2‐butanone and nonanal, emitted from dead mice are suggested as potential attractants.     

Birth of mice produced by germ cell nuclear transfer

That mammals can be cloned by nuclear transfer indicates that it is possible to reprogram the somatic cell genome to support full development. However, the developmental plasticity of germ cells is difficult to assess because genomic imprinting, which is essential for normal fetal development, is being reset at this stage. The anomalous influence of imprinting is corroborated by the poor development of mouse clones produced from primordial germ cells (PGCs) during imprinting erasure at embryonic day 11.5 or later. However, this can also be interpreted to mean that, unlike somatic cells, the genome of differentiated germ cells cannot be fully reprogrammed. We used younger PGCs (day 10.5) and eventually obtained four full-term fetuses. DNA methylation analyses showed that only embryos exhibiting normal imprinting developed to term. Thus, germ cell differentiation is not an insurmountable barrier to cloning, and imprinting status is more important than the origin of the nucleus donor cell per se as a determinant of developmental plasticity following nuclear transfer.     

Snail responses to cues produced by an invasive decapod predator

Abstract. Cues released by predators and injured prey often induce shifts in prey behavior that allow prey to evade predators, but also affect prey resource use. I investigated the effects of chemical and mechanical signals produced by injured snails (Physella gyrina) and predatory crayfish (Procambarus clarkii) on microdistributions of P. gyrina. In an initial experiment, I observed snail responses to the presence of a caged crayfish predator, to injured conspecifics, or to both. There were significant effects of time and the treatment × time interaction on the proportion of snails moving above the water line, with greater proportions of snails above the water line at night than during the day and with weak snail crawl‐out behavior being elicited by caged crayfish at night, but not during the day. In a second experiment, I examined snail microdistributions when exposed to crayfish confined to a small cage within each aquarium, crayfish confined to half of each aquarium, and crayfish ranging freely throughout each aquarium. Snails responded most strongly to free‐ranging crayfish by moving above the water line, but also demonstrated significant, but reduced, crawl‐out responses to crayfish confined to half of each aquarium; however, snails did not respond behaviorally to crayfish confined to small cages. In both experiments, there were marginally significant effects of unfed caged crayfish on the proportions of snail populations hiding under benthic shelters, with this response being the strongest at the start of the experiments but weak overall (with only 4–5% of P. gyrina responding in each experiment). These results indicate that cues (e.g., chemical or mechanical) produced by predators altered prey microdistributions, but that the exact prey responses (e.g., moving above the water line or into horizontal or benthic refugia) depended on the intensity and nature of cues.     

Cloned rabbits produced by nuclear transfer from adult somatic cells

We have developed a method to produce live somatic clones in the rabbit, one of the mammalian species considered up to now as difficult to clone. To do so, we have modified current cloning protocols proven successful in other species by taking into account both the rapid kinetics of the cell cycle of rabbit embryos and the narrow window of time for their implantation after transfer into foster recipients. Although our method still has a low level of efficiency, it has produced several clones now proven to be fertile. Our work indicates that cloning can probably be carried out successfully in any mammalian species by taking into account physiological features of their oocytes and embryos. Our results will contribute to extending the use of rabbit models for biomedical research.     

Cloned calves produced by nuclear transfer from cultured cumulus cells

Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P<0.05); that serum starvation of donor cells had no effect on blastocyst development rate of NT embryos (47.1% vs 44.4%, P>0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2–3 h, was significantly lower than that of the 3–6 h groups (31.0%), while not significantly different among 3–4 h (P < 0.05), 4–5 h, and 5–6 h groups (P≥0.05). Sixty-three thawed NT blastocysts were transferred to 31 recipient cows, of which 4 pregnancies were established and two cloned calves were given birth. These results indicate that serum starvation of cumulus cells is not a key factor for successful bovine cloning, while IFA treatment and sources of donor cells have effects on cloning efficiency.     

Immunologic mast cell-mediated responses and histamine release are attenuated by heparin.

Heparin has been shown to act as a competitive inhibitor of inositol 1,4,5-triphosphate (InsP3) receptors in various cell types. Because InsP3 is one of the second messengers involved in stimulus-secretion coupling in mast cells, it is possible that heparin may inhibit mast cell-mediated reactions. Therefore, in allergic sheep, we tested this hypothesis in two mast cell-mediated reactions induced by immunologic and nonimmunologic stimuli: immediate cutaneous reaction (ICR) and acute bronchoconstrictor response (ABR). In 12 sheep allergic to Ascaris suum antigen, the surface area of the skin wheal was determined 20 min after intradermal injection (0.05 ml) of increasing concentrations of specific antigen, compound 48/80, and histamine, without and after pretreatment with heparin (100, 300, or 1,000 U/kg i.v.). Antigen, compound 48/80, and histamine produced concentration-dependent increases in ICR. Heparin "partially" inhibited the ICR to antigen and compound 48/80 in a dose-dependent manner without modifying the ICR to histamine. The heparin preservative benzyl alcohol was ineffective. In 11 additional sheep, specific lung resistance was measured before and after inhalation challenges with antigen, compound 48/80, and histamine without and with aerosol heparin pretreatment (1,000 U/kg). Heparin blocked the antigen- and compound 48/80-induced bronchoconstriction without modifying the airway effects of histamine. In isolated human uterine mast cells, heparin inhibited the anti-immunoglobulin E- but not the calcium ionophore- (A23187) induced histamine release. These data suggest that heparin inhibits the ICR and ABR induced by stimuli that produce immunologic and nonimmunologic mast cell degranulation without attenuating the effects of histamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloned calves produced by nuclear transfer from cultured cumulus cells

Short-term cultured cumulus cell lines (1-5BCC) derived from 5 individual cows were used in nuclear transfer (NT) and 1188 enucleated bovine oocytes matured in vitro were used as nuclear recipients. A total of 931 (78.4%) cloned embryos were reconstructed, of which 763 (82%) cleaved, 627 (67.3%) developed to 8-cell stage, and 275 (29.5%) reached blastocyst stage. The average cell number of blastocysts was 124±24.5 (n=20). In this study, the effects of donor cell sources, serum starvation of donor cells, time interval from fusion to activation (IFA) were also tested on cloning efficiency. These results showed that blastocyst rates of embryos reconstructed from 5 different individuals cells were significantly different among them (14.1%, 45.2%, 27.3%, 34.3%, vs 1.5%, P<0.05); that serum starvation of donor cells had no effect on blastocyst development rate of NT embryos (47.1% vs 44.4%, P>0.05); and that blastocyst rate (20.3%) of the group with fusion/activation interval of 2-3 h, was significantly lower     

Characteristics of Korean native, Hanwoo, calves produced by transfer of in vitro produced embryos

The main objectives of this investigation were to monitor the birth weight of calves and gestation length following artificial insemination (AI) and transfer of in vivo or in vitro produced Korean native, Hanwoo embryos. Embryos produced in vivo were recovered from uterine flushings of superovulated cows 7 days after AI. Those embryos produced in vitro were co-cultured with cumulus cells for 7-8 days after in vitro fertilization. The birth weights of calves following the transfer of in vitro produced (IVP) embryos were heavier than calves from both of AI- and in vivo-derived embryo transferred calves in both sexes (29.6, 24.1 and 25.2kg, respectively, P<0.05). The IVP calves also had a longer gestation length (293.9, 285.8 and 283.8 days, respectively, P<0.05).     

Myotonic responses produced by alteration of the function of ionic channels

It has been established that in sodium isethionate Ringer's solution containing chloride ions at low concentration (0-6.1 mmol/l) a repetitive firing of action potentials (APs) could be induced systematically by stimulation of muscle fibres with single square impulses. Repetitive firings occurred also on slow depolarization elicited by different agents e.g. by some antibiotics (primycin, desertomycin) which block potassium channels, or aldehydes (acrolein). On the basis of own and literary data it is concluded that under physiological conditions a high chloride concentration in the extracellular space prevents the occurrence of repetitive firing of APs which would result from the depolarization of tubular membrane on potassium ion accumulation in the luminal fluid of T-tubules.     

Birth of viable female dogs produced by somatic cell nuclear transfer

Since the only viable cloned offspring born in dogs was a male, the purpose of the present study was to produce female puppies by somatic cell nuclear transfer (SCNT). Adult ear fibroblasts from a 2-month-old female Afghan hound were isolated and used as donor cells. In vivo-matured canine oocytes surgically collected (approximately 72h after ovulation) from the oviducts of 23 donors were used for SCNT. After removal of the cumulus cells, oocytes were enucleated, microinjected, fused with a donor cell, and activated. A total of 167 reconstructed SCNT embryos were surgically transferred (Day 0) into the oviducts of 12 recipient bitches (average 13.9 embryos/recipient, range 6-22) with spontaneous, synchronous estrous cycles. Three pregnancies were detected by ultrasonography on Day 23, maintained to term, and three healthy female puppies (520, 460, and 520g), were delivered by Caesarean section on Day 60. These puppies were phenotypically and genotypically identical to the cell donor. In conclusion, we have provided the first demonstration that female dogs can be produced by nuclear transfer of ear fibroblasts into enucleated canine oocytes.