A Method for the Electrophoretic Analysis of Proteins and RNAs of Single Cells*

SYNOPSIS A method is described for the electrophoretic analysis of proteins or RNAs from individual amebae. The method is based on fluorographic autoradiography of semi-micro polyacrylamide gels in which [³⁵S]methionine or [³H]uridine materials from single cells have been subjected to electrophoresis. The method is more sensitive and provides better resolution than previous methods for single cells. It is suitable, also, for quantitation of the separated components.     

Water Relations of the Hemiparasite Rhinanthus serotinus before and after Attachment

Growth of the hemiparasite Rhinanthus serotinus (Schönh.) Oborny was greatly stimulated after attachment of the parasite to the roots of the host plant, Hordeum vulgare L. Before attachment the hydrostatic pressure in the xylem, determined by the pressure bomb technique, was found to be lower in Rhinanthus than in the host. It increased after the formation of haustoria between host and parasite. Apparently, the water transport to Rhinanthus was facilitated. The hydrostatic pressure remained lower than that of the host, accounting for the flow of water and solutes in the direction of the parasite and indicating that there exists a resistance to water transport in the haustoria. Water and solutes were absorbed by the cells, which increased in size. The turgor pressure of the parasite rose steeply, but the osmotic potential was hardly affected.     

EVOLUTION OF HORNS IN UNGULATES: ECOLOGY AND PALEOECOLOGY

(1) The savanna ungulate faunas of the North American Miocene were broadly similar to those of present-day East Africa in terms of overall morphological and taxonomic diversity. However, the predominant ungulates of the African faunas are bovids, which possess bony horns that are primitively sexually dimorphic in their occurrence. The predominant ungulates of the North American Tertiary were equids, camelids and oreodonts, which all lacked horns. A limited number of horned ruminants were present, but these were largely Miocene immigrants from Eurasia. Horns were also absent from the large-bodied herbivores in the endemic faunas of South America and Australia. (2) The absence of horns in equids and tylopod artiodactyls is unlikely to be due to genetic insufficiency. Bony horns were present in brontotheres, which were closely related to equids, and in protoceratids, which were closely related to camelids. Nasal horns were present in one oreodont genus. (3) Studies on living ungulates show that a strong correlation exists between habitat type, feeding behaviour, social behaviour and morphology. It is possible to use the morphological remains of extinct ungulates to reconstruct the types of feeding and social behaviour, and to use the distribution of morphologies and body sizes in a community of mammals, in conjunction with geological and paleobotanical evidence, to reconstruct the type of habitat. (4) The importance of the post-Eocene climatic changes to the history of mammalian evolution is stressed. Continents at higher latitudes have become increasingly seasonal in terms of temperature and rainfall since the equable global conditions of the early Tertiary. Savanna mosaic were the predominant biome in North America by the early Miocene, and in Eurasia by the middle Miocene. Living temperate-latitude species of ungulates may not be a reliable guide for the assessment of the interrelationship between behaviour and morphology in an evolutionary perspective, as their behaviour may have been recently adapted to a habitat type that has only been in existence since the Pleistocene. (5) The primitive condition in eupecorans and protoceratids is the absence of horns, with the presence of large sabre-like canines in the males. The first horned members of these divisions had horns in the males only. Small present-day antelope, where horns may also be present in the females of the species, are probably secondarily small. (6) Horns were acquired independently in ruminant artiodactyls at least three times, and a maximum number of seven times is not unlikely. In each case, horns first appeared at a critical body weight of about 18 kg, and in correlation with a change in habitat from closed to open woodland. (7) Horns in living ruminants are associated with territorial defence by males holding exclusive feeding and reproductive territories in woodland habitats. Such behaviour in present-day antelope is correlated with a body size of greater than 15 kg and a folivorous diet. It is argued that horns evolved in ruminant artiodactyls on the adoption of this type of territorial behaviour once the critical combination of body size, diet and habitat type had been attained in their evolution from small, essentially frugivorous, forest-dwelling animals. (8) Perissodactyls never evolved sexually dimorphic bony horns of the type seen in ruminant artiodactyls. This is because their foraging and digestive strategies necessitate a larger daily intake of food. In a woodland habitat they were never able to adopt a feeding area small enough to make exclusive territory maintenance an economical proposition. Territory holding in male perissodactyls is seen, but under the opposite conditions of habitat to territorial behaviour in ruminant artiodactyls. (9) Study of the morphology and paleoecology of oreodonts suggests that they were woodland herd-forming browsers with exclusively folivorous diets. They probably had some forestomach fermentation, but did not chew the cud. Similar studies of Tertiary camelids suggest that they were predominantly selective browsers eating herbage at a low level in open country and formed mixed-sex feeding groups. These combinations of feeding and social behaviour suggest a more open structure of the mid-Tertiary habitat in North America than in Eurasia. (10) Studies of the behaviour and morphology of living members of the Ruminantia, and of the morphology and paleoecology of their fossil ancestors, suggest that they were primitively tree browsers living in closed woodland habitats. Such habitats were abundant in the Old World, but in limited supply in North America during the Oligocene, where the protoceratids were the only ungulates to parallel the eupecoran type of feeding and social behaviour. South America appears to have had an even more open habitat in the Oligocene than North America, and no parallel to the eupecorans was seen amongst the indigenous ungulates. The radiation of the Bovidae into open grassy habitats in the Pliocene may have been dependent on the immigration of grazing equids into the Old World. (11) I conclude that there was a difference in habitat structure between North America and the Old World during the Tertiary. The food resources in North America were more widely dispersed, and this may have been the result of the trees being more widely spaced. A possible causal mechanism for this was the stable land mass of the North American continent during the Tertiary, resulting in a more continental climate, with a more severe effect of the post-Eocene seasonality on the vegetation. The faunal record of the two continents also implied a greater density of trees in the Old World. (12) Thus most endemic North American ruminants did not evolve horns because, at the critical combination of body size and diet seen in the evolution of horns in the Old World ruminants, the dispersal of the food resources within the vegetation was too great for an effective home range to be maintained as an exclusive territory. (13) Attention is drawn to the dangers of constructing evolutionary stories about living animals without primary reference to the fossil record to see if the hypotheses are upheld, and of assuming that fossil animal communities can be made to fit models of existing communities.     

Correlation between leaf growth variables suggest intrinsic and early controls of leaf size in Arabidopsis thaliana

Leaf development is affected by both internal (genetic) and external (environmental) regulatory factors. The aim of this work was to investigate how leaf growth variables are related to one another in a range of environments. The leaf growth variables of wild-type Arabidopsis thaliana and leaf development mutants (ang4, ron2-1, elo1, elo2 and elo4) were studied under different incident light treatments (light and shade). The leaves studied were altered in various leaf development variables, such as the duration of expansion, relative and absolute expansion rates, epidermal cell size, epidermal cell number and initiation rate. Final leaf area was correlated to maximal absolute leaf expansion rate and cell number, but not to duration of leaf expansion or cell size. These relationships were common to all studied genotypes and light conditions, suggesting that leaf size is determined early in development. In addition, the early variables involved in leaf development were correlated to one another, and initial relative expansion rate was negatively correlated to the duration of expansion. These relationships between the leaf development variables were used to construct a conceptual model of leaf size control.     

Tribal Languages and the Challenges of Revitalization

Although school- and university-based language programs can help strengthen threatened Indigenous languages, language revitalization at its heart involves reestablishing traditional functions of language use in the context of everyday speaker interactions. The inherent dynamics of Native oral language traditions suggest the limitations of institutions in supporting critical language learning activities that are the key to successful language renewal efforts.     

Recovery of plant DNA using a reciprocating saw and silica‐based columns

The time needed for hand grinding and the cost of commercially available extraction kits remain to be the major limitations in plant DNA extraction for many researchers. We present inexpensive techniques for (i) simultaneously machine grinding large numbers of plant samples for DNA extraction using a commercially available reciprocating saw; and (ii) DNA recovery using silica column‐based extractions similar to that used in some commercially available kits. Used together, these allow for the rapid recovery of plant DNA at relatively low cost. Furthermore, these methods appear to be widely applicable within plants with good yields recovered in test extractions across major plant groups (ferns, gymnosperms, monocots and eudicots).     

Contamination of the Deep Tissues of Carcasses by Bacteria Present on the Slaughter Instruments or in the Gut

Possible routes by which bacteria might reach the deep tissues of carcasses were tested by placing genetically 'marked'strains of Escherichia coli, Clostridium perfringens or Bacillus thuringiensis on slaughter instruments before use and examining deep tissue samples for their presence post mortem . Bacteria present on the captive bolt pistol were recovered from the spleens of beef cattle and those placed on the pithing rod were found in both spleen and muscle of the flank and neck. Bacteria from the throat cutting ('stick') knife were isolated, on different occasions, from the heart, lung, spleen, liver and kidneys of sheep though rarely from their muscles. Orally administered bacteria were found in the spleen and lung, but not the musculature, of pigs.     

MISCONCEPTIONS ABOUT COERCION AND UNDUE INFLUENCE: REFLECTIONS ON THE VIEWS OF IRB MEMBERS

Payment to recruit research subjects is a common practice but raises ethical concerns relating to the potential for coercion or undue influence. We conducted the first national study of IRB members and human subjects protection professionals to explore attitudes as to whether and why payment of research participants constitutes coercion or undue influence. Upon critical evaluation of the cogency of ethical concerns regarding payment, as reflected in our survey results, we found expansive or inconsistent views about coercion and undue influence that may interfere with valuable research. In particular, respondents appear to believe that coercion and undue influence lie on a continuum; by contrast, we argue that they are wholly distinct: whereas undue influence is a cognitive distortion relating to assessment of risks and benefits, coercion is a threat of harm. Because payment is an offer, rather than a threat, payment is never coercive.     

Inactivation of thioredoxin f1 leads to decreased light activation of ADP‐glucose pyrophosphorylase and altered diurnal starch turnover in leaves of Arabidopsis plants

Chloroplast thioredoxin f (Trx f) is an important regulator of primary metabolic enzymes. However, genetic evidence for its physiological importance is largely lacking. To test the functional significance of Trx f in vivo, Arabidopsis mutants with insertions in the trx f1 gene were studied, showing a drastic decrease in Trx f leaf content. Knockout of Trx f1 led to strong attenuation in reductive light activation of ADP‐glucose pyrophosphorylase (AGPase), the key enzyme of starch synthesis, in leaves during the day and in isolated chloroplasts, while sucrose‐dependent redox activation of AGPase in darkened leaves was not affected. The decrease in light‐activation of AGPase in leaves was accompanied by a decrease in starch accumulation, an increase in sucrose levels and a decrease in starch‐to‐sucrose ratio. Analysis of metabolite levels at the end of day shows that inhibition of starch synthesis was unlikely due to shortage of substrates or changes in allosteric effectors. Metabolite profiling by gas chromatography–mass spectrometry pinpoints only a small number of metabolites affected, including sugars, organic acids and ethanolamine. Interestingly, metabolite data indicate carbon shortage in trx f1 mutant leaves at the end of night. Overall, results provide in planta evidence for the role played by Trx f in the light activation of AGPase and photosynthetic carbon partitioning in plants.