Variability in messenger RNA levels in human umbilical vein endothelial cells of different lineage and time in culture

Summary Levels of seven messenger RNA species were compared in human umbilical vein endothelial cells of different lineage and time in culture. Specifically, cells obtained from the American Type Culture Collection (ATCC) and subcultured were compared to early passage cells from cultures produced in our laboratory. Messenger RNA for tissue plasminogen activator, plaminogen activator inhibitor 1, urokinase, and thrombomodulin were expressed at higher levels in the ATCC cells. Thrombospondin, von Willebrand's Factor, and protein S messenger RNA were expressed at higher levels in the cells that we isolated. In addition, in the ATCC cells a shift in the proportion of plasminogen activator inhibitor messenger RNA from the 3.4 to the 2.4 kilobase species was found. We conclude that specific messenger RNA levels can vary considerably between cultured human umbilical vein endothelial cells. The large variation in mRNA levels which we describe has important implications for experiments involving gene expression in cultured endothelium.     

Stabilized baculovirus vector expressing a heterologous gene and GP64 from a single bicistronic transcript

The efficient scale-up of recombinant protein production in insect-cell bioreactors using baculovirus expression vectors is hampered by reductions in yield with increasing viral passage, the so-called passage effect. This phenomenon is characterized by the generation and subsequent accumulation of defective interfering baculoviruses (DIs), which interfere with the replication of genomically intact virus. A novel baculovirus expression vector is presented equipped with a bicistronic expression cassette that allows the simultaneous expression of the recombinant gene (GFP, first cistron) and an essential baculovirus gene (GP64, second cistron) from a single messenger RNA (mRNA). The translation of GP64 is mediated by an internal ribosome entry site (IRES) element from Rhopalosiphum padi virus (RhPV) while the native GP64 gene is deleted. In this way, a dominant selection pressure is placed on the entire bicistronic mRNA and hence on the maintenance of the foreign gene. The bicistronic expression vector was superior to the control baculovirus vector in that GFP expression remained at much higher levels upon continued virus passage. The versatility of this stabilized vector was demonstrated by its ability to propagate in a number of cell lines including Sf21, Sf9 and High Five cells. This novel baculovirus vector is especially valuable for large-scale recombinant protein production in insect-cell bioreactors where the number of viral passages is high.     

Human adipose-derived mesenchymal stem cells: serial passaging, doubling time and cell senescence

Adult adipose-derived mesenchymal stem cells (AD-MSC) are very interesting to our research group because they are easy to harvest, they are abundant in humans, and they have potential clinical applications in autologous cell therapy for disc degeneration. We examined these cells through sequential serial passages to assess osteogenic and chondrogenic capabilities, mean doubling time and cell senescence. Osteogenic and chondrogenic potencies were maintained through 13 passages. Mean passage doubling time increased significantly with increasing passage number. When donor age was evaluated, passages 1-4 from older donors had significantly longer doubling times compared to cells from younger donors. Passages 5-11 showed similar findings when analyzed by donor age. The mean percent senescence increased significantly with cell passaging, rising from 0% at passage 1 to 3.4% at passage 13. These novel data suggest that caution should be exercised when using AD-MSC with long passage times.     

Human adipose-derived mesenchymal stem cells: serial passaging,doubling time and cell senescence

Adult adipose-derived mesenchymal stem cells (AD-MSC) are very interesting to our research group because they are easy to harvest, they are abundant in humans, and they have potential clinical applications in autologous cell therapy for disc degeneration. We examined these cells through sequential serial passages to assess osteogenic and chondrogenic capabilities, mean doubling time and cell senescence. Osteogenic and chondrogenic potencies were maintained through 13 passages. Mean passage doubling time increased significantly with increasing passage number. When donor age was evaluated, passages 1-4 from older donors had significantly longer doubling times compared to cells from younger donors. Passages 5-11 showed similar findings when analyzed by donor age. The mean percent senescence increased significantly with cell passaging, rising from 0% at passage 1 to 3.4% at passage 13. These novel data suggest that caution should be exercised when using AD-MSC with long passage times.     

Passage time,resilience, and structure of compartmental models

The relation between the graphical structure and two measures of stability of a compartmental model is studied. The two are resilience as measured by deviation from equilibrium throughout the history of the system and mean first passage time from a central compartment back to itself. A number of examples are studied and a theory developed for models whose graphical structure is in the form of an advanced rosette. The resilience, known to increase with complexity, is shown to increase as well with uneveness in length of food chains. The mean first passage time, on the other hand, depends only on the number of food chains and their average length.     

Ecological conditions in wintering and passage areas as determinants of timing of spring migration in trans-Saharan migratory birds

1.?Climate change has been associated with shifts in the timing of biological events, including the spring arrival of migratory birds. Early arrival at breeding sites is an important life-history trait, usually associated with higher breeding success and therefore, susceptible to selection and evolution in response to changing climatic conditions. 2.?Here, we examine the effect of changes in the environmental conditions of wintering and passage areas on the mean passage time of 13 trans-Saharan passerines during their spring migration through the western Mediterranean over the 15 years from 1993 to 2007. 3.?We found that most of the species studied have been advancing the timing of their passage in recent years. However, annual variation in the mean date of passage was positively correlated with vegetation growth (measured as the normalized difference vegetation index [NDVI]) both in the Sahel (the region of departure) and in northern Africa (the passage area). Thus, migration dates were delayed in years with high primary productivity in passage and wintering zones. All species seem to respond similarly to NDVI in the Sahel; however, late migrants were less affected by ecological conditions in northern Africa than those migrating earlier, suggesting differences based on species ecology. 4.?Mean timing of passage was not related to the North Atlantic Oscillation (NAO), El Ni?o-Southern Oscillation (ENSO), temperature or NDVI in the species-specific wintering areas (the overwintering region) when analysed in combination with the other covariates. 5.?Our findings show that ecological conditions in the winter quarters (specifically the Sahel) and en route are relevant factors influencing trends in the passage dates of trans-Saharan migratory birds on the southern fringe of Europe. Possible long-term consequences for late arriving spring migrants are discussed.     

Diel periodicity and chronology of upstream migration in yellow-phase American eels (<Emphasis Type="Italic">Anguilla rostrata</Emphasis>)

Yellow-phase American eel (Anguilla rostrata) upstream migration is temporally punctuated, yet migration chronology within diel time periods is not well-understood. This study examined diel periodicity, chronology, and total length (TL) of six multi-day, high-count (285–1,868 eels) passage events of upstream migrant yellow-phase American eels at the Millville Dam eel ladder, lower Shenandoah River, West Virginia during 2011–2014. We categorized passage by diel periods (vespertine, nocturnal, matutinal, diurnal) and season (spring, summer, late summer/early fall, fall). We depicted passage counts as time-series histograms and used time-series spectral analysis (Fast Fourier Transformation) to identify cyclical patterns and diel periodicity of upstream migration. We created histograms to examine movement patterns within diel periods for each passage event and fit normal mixture models (2–9 mixtures) to describe multiple peaks of passage counts. Periodicity of movements for each passage event followed a 24-h activity cycle with mostly nocturnal movement. Multimodal models were supported by the data; most modes represented nocturnal movements, but modes at or near the transition between twilight and night were also common. We used mixed-model methodology to examine relationships among TL, diel period, and season. An additive-effects model of diel period + season was the best approximating model. A decreasing trend of mean TL occurred across diel movement periods, with the highest mean TL occurring during fall relative to similar mean values of TL for spring, summer, and late summer/early fall. This study increased our understanding of yellow-phase American eels by demonstrating the non-random nature of their upstream migration.     

Physiological salines and the mechanical properties of trout red blood cells

Suspensions of rainbow trout erythrocytes in different physiological salines were compared with respect to their haematological and filtration properties.
A method is described for the suspension of erythrocytes in Cortland saline which has proved suitable for studies of their mechanical properties over periods of several hours.
Significant differences were found between whole blood samples taken during cannulation and after several days recovery, particularly mean cell volume, frequency distribution of red cell volumes and the pore passage time through nucleopore filters. These differences were also found using red cell suspensions of the same bloods. The pore passage time of whole blood sampled during cannulation or its suspensions is less than that of recovery blood although its mean cell volume is greater.     

The control of ribonucleic acid synthesis in bacteria. Steady-state content of messenger ribonucleic acid in Escherichia coli M.R.E. 600

1. The technique of DNA-RNA hybridization was used to follow changes in the amount and average lifetime of unstable messenger RNA in Escherichia coli M.R.E. 600 over a wide range of different growth conditions. The method of analysis was based on the kinetics of incorporation of exogenous labelled nucleic acid bases into the RNA of steadily growing cultures, as described by Bolton & McCarthy (1962). 2. The ratio of the average lifetime of messenger RNA to the mean generation time of E. coli cultures was constant over the temperature range 25-45 degrees C in a given medium, but the constant varied with the nature of the growth medium. For cultures growing in sodium lactate-salts or glucose-salts media the ratio was 0.046+/-0.005 and in enriched broth it was 0.087+/-0.009. Measurements of the amounts of transfer RNA, ribosomal RNA and messenger RNA were also made. The results confirmed earlier reports that the ratio of the amount of messenger RNA to the amount of ribosomes in the cells is virtually constant. On the other hand, the ratio of the amount of transfer RNA to the amount of ribosomal RNA decreased with increasing growth rate at a given temperature. 3. In cultures at temperatures higher than necessary for optimum rates of growth the average lifetime of messenger RNA lengthened in harmony with the increased time required for cell division. It seems that suboptimum growth rates at higher temperatures cannot be explained simply as a combination of increased rates of synthesis and breakdown of messenger RNA with a grossly decreased efficiency of translation. The absolute rate of messenger RNA synthesis was lowered, and its amount in the cells was typical of all other cultures grown at lower temperatures in the same medium. 4. The rate of entry of exogenous labelled uracil into unstable messenger RNA and stable ribosomal RNA was constant in all media at all temperatures in the approximate ratio 1:2. In media supporting a lower rate of growth, e.g. lactate-salts or glucose-salts media, the messenger RNA fraction constituted 2.2+/-0.3% of the total cellular RNA. In enriched broth 3.6+/-0.3% of the total RNA was messenger.     

Substance P modulates single-channel properties of neuronal nicotinic acetylcholine receptors.

Substance P (SP) is present in avian sympathetic ganglia and accelerates the decay rate of acetylcholine (ACh)-evoked macroscopic currents in sympathetic neurons. We demonstrate here that SP modulates ACh-elicited single channels in a manner consistent with an enhancement of ACh receptor (AChR) desensitization. Furthermore, since AChR channel function was monitored in cell-attached patches with SP applied to the extra-patch membrane, the peptide must act via a second messenger mechanism. SP specifically decreases the net ACh-activated single-channel current across the patch membrane by decreasing both channel opening frequency and mean open time kinetics. These experiments demonstrate that a peptide can modulate neuronal AChR function by a second messenger mechanism.     

The relationship of food intake and ingesta passage predicts feeding ecology in two different megaherbivore groups

Digestion, especially of plant material, is a time-dependent process. In herbivores, an increase in food intake is usually correlated to an acceleration of ingesta passage through the gut, and could hence depress digestive efficiency. Therefore, the nature of the relationship between food intake and ingesta passage (i.e. whether the increase in ingesta passage due to the increase in food intake is mild or drastic) should determine the flexibility of the feeding strategy of herbivore and omnivore species. Using two megaherbivore groups, the elephants and the hippopotamuses, as examples from opposing ends of the range of potential adaptations to this problem, we demonstrate that the species-specific relationship of food intake and ingesta passage can precisely predict feeding ecology and activity budgets. In hippos, the distinct acceleration in ingesta passage due to increased intake limits the additional energy gained from eating more forage, and explains the comparatively low food intake and short feeding times generally observed in these animals. In elephants, increased food intake only leads to a very moderate increase of ingesta passage, thus theoretically allowing to optimize energy gain by eating more, which is in accord with the high food intake and long feeding times observed in these animals. We suggest that the characterization of the intake-passage relationship in herbi- and omnivorous species is of much higher ecological relevance than the determination of a supposedly species-specific "passage time/mean retention time".     

KINETIC AND HISTOLOGICAL CHANGES OF A SERIALLY TRANSPLANTED MOUSE TUMOUR

A serially transplanted mouse tumour, NT1, was studied histologically and by autoradiography using tritiated thymidine at three stages in its transplant history: after two, seventeen and twenty-seven passages. the growth rate of the tumour decreased progressively with increasing passage number, and the mean cycle time of the tumour cells increased from 21 hr to 34 hr between the seventeenth and twenty-seventh passages. Histologically the tumour changed from having an epithelial structure (second passage), to having a mixed structure with at least two histologically different regions (seventeenth passage), to having a fibrous structure (twenty-seventh passage). Radiation response experiments were carried out on the second and twenty-seventh passaged tumours, the results of which are consistent with the kinetic and histological changes.     

The diffusion transit time; A simple derivation

The mean first passage time for free diffusion can be derived directly by solving a simple analogue steady state problem. In this problem the diffusion starting region is considered as a time independent source of diffusing particles and the diffusion target assumes the behaviour of a perfectly absorbing sink. It is shown here that the transit time between the source and the sink, which in this particular problem is equal to the ratio between the holdup of the system and the total flux, is identical to the Brownian movement concept of the mean first passage time for free diffusion. This established identity considerably facilitates the derivation and investigation of the timing of diffusion in complicated structures such as those commonly found in living organisms.     

Blackcaps Sylvia atricapilla stopping over at the desert edge; inter- and intra-sexual differences in spring and autumn migration

Spring and autumn Palaearctic-African migration patterns of Blackcaps Sylvia atricapilla during stopover at Elat, Israel, showed that males appeared significantly earlier than females during spring but not during autumn migration, suggesting that in males there is a stronger drive to reaching breeding territories early. The difference in mean appearance dates between sexes in spring tended to be greatest in years when the males appeared earliest. Longer spread of passage (the dates between which the central 50% of individuals were captured) for each sex in spring was found in years with an early mean passage datebut was significant only for females. These observations suggest that the timing of Blackcap migration is governed not only by endogenous factors but also by exogenous factors, and when the environmental conditions are unfavourable, the differences in passage dates between sexes decrease and the passage lengths shorten. The early individuals (both males and females) that stopped over at Elat in spring were those with relatively small body size (as indicated by relatively short wings) and relatively large fat reserves and in good body condition (as indicated from fat score and body mass/wing-length ratio). No differences in body size between early and late transients were detected during the autumn migration, but late birds of both sexes carried larger fat reserves. These phenomena may be explained either by leap-frog migration or by differential fitness among wintering males and females or both, with only the fittest Blackcaps being capable of an early departure. These individuals probably face much less intensive intra- and interspecific competition with residents and other transients in stopover sites than do later transients.     

Rapid aquaporin translocation regulates cellular water flow: mechanism of hypotonicity-induced subcellular localization of aquaporin 1 water channel

The control of cellular water flow is mediated by the aquaporin (AQP) family of membrane proteins. The structural features of the family and the mechanism of selective water passage through the AQP pore are established, but there remains a gap in our knowledge of how water transport is regulated. Two broad possibilities exist. One is controlling the passage of water through the AQP pore, but this only has been observed as a phenomenon in some plant and microbial AQPs. An alternative is controlling the number of AQPs in the cell membrane. Here, we describe a novel pathway in mammalian cells whereby a hypotonic stimulus directly induces intracellular calcium elevations through transient receptor potential channels, which trigger AQP1 translocation. This translocation, which has a direct role in cell volume regulation, occurs within 30 s and is dependent on calmodulin activation and phosphorylation of AQP1 at two threonine residues by protein kinase C. This direct mechanism provides a rationale for the changes in water transport that are required in response to constantly changing local cellular water availability. Moreover, because calcium is a pluripotent and ubiquitous second messenger in biological systems, the discovery of its role in the regulation of AQP translocation has ramifications for diverse physiological and pathophysiological processes, as well as providing an explanation for the rapid regulation of water flow that is necessary for cell homeostasis.     

Structural features of the tmRNA–ribosome interaction

Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA–ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.