Optimization of porcine urothelial cell cultures: Best practices,recommendations, and threats

Many experimental approaches have been conducted in order to isolate urothelial cells from bladder tissue biopsies, but each method described has utilized different protocols and sources of bladder tissue. In this study, we compared the different methods of urothelial cell isolation available in literature together with standardized methods in order to obtain more unified results. Five methods for primary porcine urothelial culture establishment were compared: tissue explants and four enzymatic methods utilizing collagenase II, dispase II, combination of dispase II and trypsin, and trypsin alone. The average number of isolated cells, cell morphology, success of established culture, average number of cells from the first passage, expression of p63 and pancytokeratin and the characterization of urothelial cell growth, and aging were analyzed during the in vitro culture. The method utilizing dispase II was the most efficient and reproducible method for the isolation and culture of porcine urothelial cells when compared to the other tested methods. Urothelial cells obtained by this method grew considerably well and the cultures were established with high efficiency, which enabled us in obtaining a large quantity of cells with normal morphology. Contamination with fibroblasts in this method was the lowest. The utilization of a proper method for urothelial cell isolation is a critical step in the urinary tract regeneration when using tissue engineering techniques. In summary, this study demonstrated that by utilizing the described method with dispase II, a suitable number of cells was achieved, proving the method useful for tissue regeneration.     

N18-RE-105 cells: differentiation and activation of p53 in response to glutamate and adriamycin is blocked by SV40 large T antigen tsA58

Process extension was induced in cells of the N18-RE-105 neuroblastoma-retinal hybrid line by toxic agents, including glutamate and the p53-inducing anticancer agents adriamycin and etoposide. Both adriamycin and glutamate activated p53 as measured by a plasmid transfection assay. It was therefore hypothesized that SV40 large T antigen, which binds p53, would interfere with cellular differentiation. To test this hypothesis, the temperature-sensitive form of SV40 large T was transduced into N18-RE-105 cells by retroviral infection. SV40 large T-infected cells became de-differentiated, grew in tightly-packed colonies, lost expression of neurofilament, and lost the ability to differentiate in response to glutamate and adriamycin. The de-differentiating effect of SV40 large T antigen may be due to binding and inactivation of cellular proteins, such as p53, p107, p130, p300, and retinoblastoma protein, which are important in cellular growth and differentiation. It is suggested that p53 may play a role in cellular differentiation, perhaps under unusual circumstances involving stress or cytotoxicity. Received: 29 April 1997 / Accepted: 18 June 1997     

Endocrine background of how 20-hydroxyecdysone agonist, RH 5849, influences diurnal pattern of pupation in shape Spodoptera littoralis

Under laboratory conditions (L16:D8, 24°C, r.h. 80%), applying of an ecdysone agonist, RH 5849, (1,2-dibenzoyl-1-tert-butylhydrazine) onto final instar larvae of Spodoptera littoralis (Boisd.), Noctuidae, Lepidoptera, delayed or advanced the time of pupation depending on the time of treatment. This response was dose-dependent. In addition, non-lethal doses of RH 5849, ranging from 0.05 to 10 µg/larva were used. Following treatment of S. littoralis larvae with 5 µg RH 5849 the endogenous ecdysteroid level was increased or decreased depending on the time of application. Based on the results presented, we suggest that potential users of non-steroidal ecdysone agonists should consider the proper circadian timing of application in the field.     

Tracermer signal generators: An arborescent approach to the incorporation of multiple chemiluminescent labels

The synthesis, conjugation, and chemiluminescent evaluation of zero, first, and second order acridinium-based Tracermer^TM signal generators are described. Members of this family of labels have potential use as tracers in diagnostic assays and are structurally similar to arborol dendrimers. Tracermer^TM-BSA conjugates showed up to a sixfold increase in light emission compared to the normal acridinium label.     

Basicranial flexion,relative brain size,and facial kyphosis in Homo sapiens and some fossil hominids

Comparative work among nonhominid primates has demonstrated that the basicranium becomes more flexed with increasing brain size relative to basicranial length and as the -upper and lower face become more ventrally deflected (Ross and Ravosa [1993] Am. J. Phys. Anthropol. 91:305–324). In order to determine whether modern humans and fossil hominids follow these trends, the cranial base angle (measure of basicranial flexion), angle of facial kyphosis, and angle of orbital axis orientation were measured from computed tomography (CT) scans of fossil hominids (Sts 5, MLD 37/38, OH9, Kabwe) and lateral radiographs of 99 extant humans. Brain size relative to basicranial length was calculated from measures of neurocranial volume and basicranial length taken from original skulls, radiographs, CT scans, and the literature. Results of bivariate correlation analyses revealed that among modern humans basicranial flexion and brain size/basicranial length are not significantly correlated, nor are the angles of orbital axis orientation and facial kyphosis. However, basicranial flexion and orbit orientation are significantly positively correlated among the humans sampled, as are basicranial flexion and the angle of facial kyphosis. Relative to the comparative sample from Ross and Ravosa (1993), all hominids have more flexed basicrania than other primates: Archaic Homo sapiens, Homo erectus, and Australopithecus africanus do not differ significantly from Modern Homo sapiens in their degree of basicranial flexion, although they differ widely in their relative brain size. Comparison of the hominid values with those predicted by the nonhominid reduced major-axis equations reveal that, for their brain size/basicranial length, Archaic and Modern Homo sapiens have less flexed basicrania than predicted. H. erectus and A. africanus have the degree of basicranial flexion predicted by the nonhominid reduced major-axis equation. Modern humans have more ventrally deflected orbits than all other primates and, for their degree of basicranial flexion, have more ventrally deflected orbits than predicted by the regression equations for hominoids. All hominoids have more ventrally deflected orbital axes relative to their palate orientation than other primates. It is argued that hominids do not strictly obey the trend for basicranial flexion to increase with increasing relative brain size because of constraints on the amount of flexion that do not allow it to decrease much below 90°. Therefore, if basicranial flexion is a mechanism for accommodating an expanding brain among non-hominid primates, other mechanisms must be at work among hominids. © 1995 Wiley-Liss, Inc.     

Climate and microhabitat shape the prevalence of endozoochory in the seed rain of tropical montane forests

Endozoochory, the dispersal of seeds by animal ingestion, is the most dominant mode of seed dispersal in tropical forests and is a key process shaping current and future forest dynamics. However, it remains largely unknown how endozoochory is associated with environmental conditions at regional and local scales. Here, we investigated the effects of elevation, climate, and microhabitat conditions on the proportion of endozoochorous plant species in the seed rain of the tropical Andes of southern Ecuador. Over 1 year, we measured seed rain in 162 seed traps on nine 1-ha forest plots located at 1000, 2000, and 3000 m a.s.l. We recorded climatic conditions (mean annual temperature and rainfall) in each plot and microhabitat conditions (leaf area index and soil moisture) adjacent to each seed trap. In total, we recorded 331,838 seeds belonging to 323 morphospecies. Overall, the proportion of endozoochorous species in the seed rain decreased with elevation. The relative biomass of endozoochorous species decreased with increasing rainfall, whereas the relative seed richness of endozoochorous species increased with increasing temperature and leaf area index. These findings suggest an interplay between climate factors and microhabitat conditions in shaping the importance of endozoochorous plant species in the seed rain of tropical montane forests. We conclude that changing climatic and microhabitat conditions are likely to cause changes in the dominant dispersal modes of plant communities which may trigger changes in the current and future dynamics of tropical forests.     

Egg predation by copepods in Daphnia brood cavities

Both field observations and enclosure experiments show that juvenile copepods enter Daphnia brood cavities to feed on Daphnia eggs and embryos. The ability to perform such in vivo exploitation is reported for both cyclopoid and calanoid copepods. Copepodites of Acanthocyclops robustus (G.O. Sars) were found to eat eggs in brood cavities of D. magna, D. pulex and D. pulicaria in experimental enclosures rich in algae. Copepodites of Eudiaptomus gracilus (G.O. Sars) were found in brood cavities of D. hyalina in a mesotrophic lake. The copepods' intrusions into brood cavities caused dramatic declines in the clutch size of infested Daphnia, and this predation effect could easily be confused with the effect of severe food limitation.     

Seasonal changes in light transmission by bud scales of spruce and pine

A bundle of optical fibres and a photometer were used to measure light transmission through bud scales of Norway spruce [Picea abies (L.) Karst.] and Scots pine (Pinus silvestris L.). Percentage transmission increases with wavelength from none below 480 nm to 3% the far-red. Transmission of all wavebands measured was lowest in late summer and winter and highest in the autumn and spring. For most of the year in pine, and only from July to September in spruce, there is a depression of red light (660–670 nm) transmission. This affects the ratio of 660/730 nm light penetrating the apical domes. This ratio is lowest in July and August, at the time of initiation of female strobili. The seasonal variation in transmission of light by bud scales is discussed in relation to the possible consequences for the control of flowering.     

Light conditions inside developing buds affect floral induction

Buds of Pinus sylvestris L. and Picea abies (L.) Karst. were pierced with optical fibres allowing natural light to the vicinity of apical domes. Induction of female strobiles was achieved. Since bud scales absorb more red than far-red light, the treatment may have increased the far-red light absorbing form of phytochrome.Abbreviations Pfr far-red light absorbing form of phytochrome     

Family planning inDaphnia: resistance to starvation in offspring born to mothers grown at different food levels

Summary We observed a shift in maternal investment per offspring in clonal cultures of twoDaphnia species. Mothers grown at high food levels produced large clutches of smaller eggs but their offspring could not survive long under starvation conditions. Genetically identical mothers grown at low food levels produced small cultches of larger eggs, and their offspring, albeit low in numbers, were able to survive long periods of starvation. Our data show thatDaphnia mothers are capable of assessing food level and use this information in adjusting their fractional peroffspring allocation of reproductive resources.