Energetic costs of mange in wolves estimated from infrared thermography.

Parasites, by definition, extract energy from their hosts and thus affect trophic and food web dynamics even when the parasite may have limited effects on host population size. We studied the energetic costs of mange (Sarcoptes scabiei) in wolves (Canis lupus) using thermal cameras to estimate heat losses associated with compromised insulation during the winter. We combined the field data of known, naturally infected wolves with a data set on captive wolves with shaved patches of fur as a positive control to simulate mange-induced hair loss. We predict that during the winter in Montana, more severe mange infection increases heat loss by around 5.2-12 MJ per night (1,240-2,850 kcal, or a 65-78% increase) for small and large wolves, respectively, accounting for wind effects. To maintain body temperature would require a significant proportion of a healthy wolf's total daily energy demands (18-22 MJ/day). We also predict how these thermal costs may increase in colder climates by comparing our predictions in Bozeman, Montana to those from a place with lower ambient temperatures (Fairbanks, Alaska). Contrary to our expectations, the 14°C differential between these regions was not as important as the potential differences in wind speed. These large increases in energetic demands can be mitigated by either increasing consumption rates or decreasing other energy demands. Data from GPS-collared wolves indicated that healthy wolves move, on average, 17 km per day, which was reduced by 1.5, 1.8, and 6.5 km for light, medium, and severe hair loss. In addition, the wolf with the most hair loss was less active at night and more active during the day, which is the converse of the movement patterns of healthy wolves. At the individual level, mange infections create significant energy demands and altered behavioral patterns, this may have cascading effects on prey consumption rates, food web dynamics, predator-prey interactions, and scavenger communities.

Digestive physiology, metabolism and methane production of captive Linné's two-toed sloths (Choloepus didactylus).

Sloths are renowned for their low metabolic rate, low food intake and low defecation frequency. We investigated factors of digestive physiology and energy metabolism in four captive individuals (mean body mass 10.0 ± SD 3.7 kg) of a hitherto mostly unstudied sloth species, Linné's two-toed sloth (Choloepus didactylus), in a 2-week digestion recording and 23-h respiration experiment on animals fed a standard zoo diet of vegetables and starchy components. Dry matter intake, defecation frequency and particle mean retention time (MRT) in the gastrointestinal tract (GIT) were 12 ± 3 g/(kg(0.75)  day), once every 5 days and >140 h in three individuals, but 53 g/(kg(0.75)  day), daily and 82 h in one individual that was apparently compensating for a period of weight loss prior to the experiment. In all animals, solute marker was eliminated at a faster rate than the particle marker, indicating 'digesta washing' in the sloths' GIT. The overall metabolic rate calculated from oxygen consumption matched the metabolisable energy intake in three individuals [173 ± 22 vs. 168 ± 44 kJ/(kg(0.75)  day)] but not in the fourth one [225 vs. 698 kJ/(kg(0.75)  day)], supporting the interpretation that this animal was replenishing body stores. In spite of the low food intake and the low-fibre diet (209 ± 26 g neutral detergent fibre/kg dry matter), methane production was rather high accounting for 9.4 ± 0.8% of gross energy intake (2.7% in the fourth individual), which exceeded literature data for ruminants on forage-only diets. These results corroborate literature reports on low intake, low defecation frequency, low metabolic rate and long MRT in other sloth species. The long MRT is probably responsible for the comparatively high methane production, providing more opportunity for methanogenic archaea than in other non-ruminant mammals to produce significant amounts of methane.

Environmental osmolality influences sperm motility activation in an anuran amphibian.

Evolutionary theory predicts that selection will favour sperm traits that maximize fertilization success in local fertilization environments. In externally fertilizing species, osmolality of the fertilization medium is known to play a critical role in activating sperm motility, but there remains limited evidence for adaptive responses to local osmotic environments. In this study, we used a split-sample experimental design and computer-assisted sperm analysis to (i) determine the optimal medium osmolality for sperm activation (% sperm motility and sperm velocity) in male common eastern froglets (Crinia signifera), (ii) test for among-population variation in percentage sperm motility and sperm velocity at various activation-medium osmolalities and (iii) test for among-population covariation between sperm performance and environmental osmolality. Frogs were obtained from nine populations that differed in environmental osmolality, and sperm samples of males from different populations were subjected to a range of activation-medium osmolalities. Percentage sperm motility was optimal between 10 and 50 mOsm kg(-1) , and sperm velocity was optimal between 10 and 100 mOsm kg(-1) , indicating that C. signifera has evolved sperm that can function across a broad range of osmolalities. As predicted, there was significant among-population variation in sperm performance. Furthermore, there was a significant interaction between activation-medium osmolality and environmental osmolality, indicating that frogs from populations with higher environmental osmolality produced sperm that performed better at higher osmolalities in vitro. This finding may reflect phenotypic plasticity in sperm functioning, or genetic divergence resulting from spatial variation in the strength of directional selection. Both of these explanations are consistent with evolutionary theory, providing some of the first empirical evidence that local osmotic environments can favour adaptive sperm motility responses in species that use an external mode of fertilization.

The effects of maternal calcium status during late gestation on lamb growth and survival in twin-bearing Merino ewes grazing pasture.

Our project aimed to investigate whether low maternal calcium status during late gestation decreased weight, growth and survival of lambs born to twin-bearing Merino ewes grazing pasture. On day 130 of gestation, approximately a 10 mL blood sample was collected from 108 twin-bearing Merino ewes. Blood pH and calcium ion concentration were measured in whole blood and serum was analysed for calcium, magnesium, phosphate, beta hydroxy butyrate, non-esterified fatty acids and cholesterol. Six to 18 h after birth and at 49 days of age, lamb live weight was recorded. Ewe serum calcium level at day 130 of gestation were divided into tertiles (1.58-2.13, 2.14-2.30, and 2.31-2.99 mmol/L) with ewes classified as either Low, Med or High (n = 36 per group). A general linear mixed model was used to analyse the data. Survival to 1, 2, 3 and 7 days of age was lower for lambs born to ewes in the Low calcium group compared with those born in the Med and High calcium groups (P < 0.05). At day 49 of age, lamb survival tended to be lower for lambs in the Low (73.6%) compared with Med (86.8%) and High (85.3%) calcium groups (P < 0.1). There were no differences in lamb body weight (P > 0.05). Calcium levels of ewes in the Low calcium group were, on average, indicative of subclinical hypocalcaemia, with this group of ewes also having the lowest lamb survival rate, suggesting that maternal subclinical hypocalcaemia reduces lamb survival. Further studies, particularly on improving maternal calcium levels, are warranted.

Potential roles for prions and protein-only inheritance in cancer.

Inherited mutations are known to cause familial cancers. However, the cause of sporadic cancers, which likely represent the majority of cancers, is yet to be elucidated. Sporadic cancers contain somatic mutations (including oncogenic mutations); however, the origin of these mutations is unclear. An intriguing possibility is that a stable alteration occurs in somatic cells prior to oncogenic mutations and promotes the subsequent accumulation of oncogenic mutations. This review explores the possible role of prions and protein-only inheritance in cancer. Genetic studies using lower eukaryotes, primarily yeast, have identified a large number of proteins as prions that confer dominant phenotypes with cytoplasmic (non-Mendelian) inheritance. Many of these have mammalian functional homologs. The human prion protein (PrP) is known to cause neurodegenerative diseases and has now been found to be upregulated in multiple cancers. PrP expression in cancer cells contributes to cancer progression and resistance to various cancer therapies. Epigenetic changes in the gene expression and hyperactivation of MAP kinase signaling, processes that in lower eukaryotes are affected by prions, play important roles in oncogenesis in humans. Prion phenomena in yeast appear to be influenced by stresses, and there is considerable evidence of the association of some amyloids with biologically positive functions. This suggests that if protein-only somatic inheritance exists in mammalian cells, it might contribute to cancer phenotypes. Here, we highlight evidence in the literature for an involvement of prion or prion-like mechanisms in cancer and how they may in the future be viewed as diagnostic markers and potential therapeutic targets.

No effect of short-term exposure to high-fibre diets on the gastrointestinal morphology of layer hens (Gallus gallus domesticus): body reserves are used to manage energy deficits in favour of phenotypic plasticity.

Using layer hens, Gallus gallus domesticus, we compared the digestive capabilities of birds on a low-fibre diet (LF, 8.49% neutral detergent fibre; NDF), with those fed a high-fibre diet balanced for energy and protein to match the LF diet (high fibre balanced, HFB; NDF = 15.61%) and those fed a high fibre unbalanced (HFU) diet (NDF = 16.68%). The HFU diet had the lowest apparent dry matter (DM) metabolisability at 58.14 ± 6.46%, followed by HFB, 65.87 ± 3.50 and the LF diet, 70.49 ± 7.07%. Despite significant differences between apparent DM metabolisabilities of LF and HFU diets, no morphometric changes in the gastrointestinal tract (GIT) of layer hens were observed (including crop, gizzard, proventriculus, liver, large intestine, paired caeca and small intestine). Conversely, body mass losses were recorded for animals on HFU diet, while those on the LF and HFB diets actually gained body mass over the 14-day trials. We suggest that the body mass losses seen in the animals fed HFU diets were attributed to losses in adipose tissue, but this was not quantified. Assuming body mass losses were mainly in adipose tissue, we propose that adipose may act to buffer environmental challenges like shortfalls in nutrient acquisition when dietary energy requirements are not met. Compared with smaller birds (e.g. quail), the larger body size of the layer hens may offer them a greater safety margin in terms of body energy reserves before changes in the GIT might be needed to redress energy deficits associated with hard-to-digest, high-fibre diets.

Energy and water use by invasive goats (Capra hircus) in an Australian rangeland, and a caution against using broad-scale allometry to predict species-specific requirements.

Feral goats (Capra hircus) are ubiquitous across much of Australia's arid and semi-arid rangelands, where they compete with domestic stock, contribute to grazing pressure on fragile ecosystems, and have been implicated in the decline of several native marsupial herbivores. Understanding the success of feral goats in Australia may provide insights into management strategies for this and other invasive herbivores. It has been suggested that frugal use of energy and water contributes to the success of feral goats in Australia, but data on the energy and water use of free-ranging animals are lacking. We measured the field metabolic rate and water turnover rate of pregnant and non-pregnant feral goats in an Australian rangeland during late summer (dry season). Field metabolic rate of pregnant goats (601 ± 37 kJ kg(-0.73)d(-1)) was 1.3 times that of non-pregnant goats (456 ± 24 kJ kg(-0.73)d(-1)). The water turnover rate of pregnant goats (228 ± 18 mL kg(-0.79)d(-1)) was also 1.3 times that of non-pregnant goats (173 ± 18 kg(-0.79)d(-1)), but the difference was not significant (P=0.07). There was no significant difference in estimated dry matter digestibility between pregnant and non-pregnant goats (mean ca. 58%), blood or urine osmolality, or urine electrolyte concentrations, indicating they were probably eating similar diets and were able to maintain osmohomeostasis. Overall, the metabolic and hygric physiology of non-pregnant goats conformed statistically to the predictions for non-marine, non-reproductive placental mammals according to both conventional and phylogenetically independent analyses. That was despite the field metabolic rate and estimated dry matter intake of non-pregnant goats being only 60% of the predicted level. We suggest that general allometric analyses predict the range of adaptive possibilities for mammals, but that specific adaptations, as present in goats, result in ecologically significant departures from the average allometric curve. In the case of goats in the arid Australian rangelands, predictions from the allometric regression would overestimate their grazing pressure by about 40% with implications for the predicted impact on their local ecology.

Review: Comparative methane production in mammalian herbivores.

Methane (CH4) production is a ubiquitous, apparently unavoidable side effect of fermentative fibre digestion by symbiotic microbiota in mammalian herbivores. Here, a data compilation is presented of in vivo CH4 measurements in individuals of 37 mammalian herbivore species fed forage-only diets, from the literature and from hitherto unpublished measurements. In contrast to previous claims, absolute CH4 emissions scaled linearly to DM intake, and CH4 yields (per DM or gross energy intake) did not vary significantly with body mass. CH4 physiology hence cannot be construed to represent an intrinsic ruminant or herbivore body size limitation. The dataset does not support traditional dichotomies of CH4 emission intensity between ruminants and nonruminants, or between foregut and hindgut fermenters. Several rodent hindgut fermenters and nonruminant foregut fermenters emit CH4 of a magnitude as high as ruminants of similar size, intake level, digesta retention or gut capacity. By contrast, equids, macropods (kangaroos) and rabbits produce few CH4 and have low CH4 : CO2 ratios for their size, intake level, digesta retention or gut capacity, ruling out these factors as explanation for interspecific variation. These findings lead to the conclusion that still unidentified host-specific factors other than digesta retention characteristics, or the presence of rumination or a foregut, influence CH4 production. Measurements of CH4 yield per digested fibre indicate that the amount of CH4 produced during fibre digestion varies not only across but also within species, possibly pointing towards variation in microbiota functionality. Recent findings on the genetic control of microbiome composition, including methanogens, raise the question about the benefits methanogens provide for many (but apparently not to the same extent for all) species, which possibly prevented the evolution of the hosting of low-methanogenic microbiota across mammals.

Endocytosis is required for the growth of vacuolar H(+)-ATPase-defective yeast: identification of six new END genes.

Yeast mutants that are defective in acidification of the lysosome-like vacuole are able to grow at pH 5.5, but not at pH 7. Here, we present evidence that endocytosis is required for this low pH-dependent growth and use this observation to develop a screen for mutants defective in endocytosis. By isolating mutants that cannot grow when they lack the 60-kD vacuolar ATPase subunit (encoded by the VAT2 gene), we isolated a number of vat2-synthetic lethal (Vsl-) mutant strains. Seven of the Vsl- mutants are defective in endocytosis. Four of these mutant strains (end8-1, end9-1, end10-1, and end11-1) show altered uptake of the endocytosed ligand, alpha-factor, and three (end12-1, end12-2, and end13-1) are probably defective in transfer of internalized material to the vacuole. Most of the mutations also confer a strong Ts- growth defect. The mutants defective in uptake of alpha-factor sort newly synthesized vacuolar proteins correctly, while those which may be defective in subsequent transport steps secrete at least a fraction of the newly synthesized soluble vacuolar proteins. The mutations that result in a defect in alpha-factor uptake are not allelic to any of the genes previously shown to encode endocytic functions.

The WASp homologue Las17p functions with the WIP homologue End5p/verprolin and is essential for endocytosis in yeast.

Several end mutations that block the internalisation step of endocytosis in Saccharomyces cerevisiae also affect the cortical actin cytoskeleton [1]. END5 encodes a proline-rich protein (End5p or verprolin) required for a polarised cortical actin cytoskeleton and endocytosis [2,3]. End5p interacts with actin [4], but its exact function is not yet known. To help elucidate End5p function, we sought other End5p-interacting proteins and identified the LAS17/BEE1 gene (encoding the yeast homologue of the human Wiskott-Aldrich Syndrome protein, WASp) as a high-copy-number suppressor of the temperature-sensitive growth and endocytic defects of end5-1 cells (carrying a frameshift mutation affecting the last 213 residues of End5p). LAS17 is unable to suppress a full deletion of END5 (end5 delta), however, suggesting that the defective End5-1p in end5-1 mutants may be stabilised by Las17p. The amino terminus of Las17p interacts with the carboxyl terminus of End5p in the yeast two-hybrid system and similar interactions have been shown between WASp and a mammalian End5p homologue, WASp-interacting protein (WIP) [5]. As las17 delta deletion mutants are blocked in endocytosis, we conclude that Las17p and End5p interact and are essential for endocytosis.

EH domain proteins Pan1p and End3p are components of a complex that plays a dual role in organization of the cortical actin cytoskeleton and endocytosis in Saccharomyces cerevisiae.

Several proteins from diverse organisms have been shown to share a region of sequence homology with the mammalian epidermal growth factor receptor tyrosine kinase substrate Eps15. Included in this new protein family, termed EH domain proteins, are two yeast proteins, Pan1p and End3p. We have shown previously that Pan1p is required for normal organization of the actin cytoskeleton and that it associates with the actin patches on the cell cortex. End3p has been shown by others to be an important factor in the process of endocytosis. End3p is also known to be required for the organization of the actin cytoskeleton. Here we report that Pan1p and End3p act as a complex in vivo. Using the pan1-4 mutant which we isolated and characterized previously, the END3 gene was identified as a suppressor of pan1-4 when overexpressed. Suppression of the pan1-4 mutation by multicopy END3 required the presence of the mutant Pan1p protein. Coimmunoprecipitation and two-hybrid protein interaction experiments indicated that Pan1p and End3p associate with each other. The localization of Pan1p to the cortical actin cytoskeleton became weakened in the end3 mutant at the permissive temperature and undetectable at the restrictive temperature, suggesting that End3p may be important for proper localization of Pan1p to the cortical actin cytoskeleton. The finding that the pan1-4 mutant was defective in endocytosis as severely as the end3 mutant under nonpermissive conditions supports the notion that the association between Pan1p and End3p is of physiological relevance. Together with results of earlier reports, these results provide strong evidence suggesting that Pan1p and End3p are the components of a complex that has essential functions in both the organization of cell membrane-associated actin cytoskeleton and the process of endocytosis.

Viability of clathrin heavy-chain-deficient Saccharomyces cerevisiae is compromised by mutations at numerous loci: implications for the suppression hypothesis.

The gene encoding clathrin heavy chain in Saccharomyces cerevisiae (CHC1) is not essential for growth in most laboratory strains tested. However, in certain genetic backgrounds, a deletion of CHC1 (chc1) results in cell death. Lethality in these chc1 strains is determined by a locus designated SCD1 (suppressor of clathrin deficiency) which is unlinked to CHC1 (S. K. Lemmon and E. W. Jones, Science 238:504-509, 1987). The lethal allele of SCD1 has no effect on cell growth when the wild-type version of CHC1 is present. This result led to the proposal that most yeast strains are viable in the absence of clathrin heavy chain because they possess the SCD1 suppressor. Discovery of another yeast strain that cannot grow without clathrin heavy chain has allowed us to perform a genetic test of the suppressor hypothesis. Genetic crosses show that clathrin-deficient lethality in the latter strain is conferred by a single genetic locus (termed CDL1, for clathrin-deficient lethality). By constructing strains in which CHC1 expression is regulated by the GAL10 promoter, we demonstrate that the lethal alleles of SCD1 and CDL1 are recessive. In both cases, very low expression of CHC1 can allow cells to escape from lethality. Genetic complementation and segregation analyses indicate that CDL1 and SCD1 are distinct genes. The lethal CDL1 allele does not cause a defect in the secretory pathway of either wild-type or clathrin heavy-chain-deficient yeast. A systematic screen to identify mutants unable to grow in the absence of clathrin heavy chain uncovered numerous genes similar to SCD1 and CDL1. These findings argue against the idea that viability of chc1 cells is due to genetic suppression, since this hypothesis would require the existence of a large number of unlinked genes, all of which are required for suppression. Instead, lethality appears to be a common, nonspecific occurrence when a second-site mutation arises in a strain whose cell growth is already severely compromised by the lack of clathrin heavy chain.

end5, end6, and end7: mutations that cause actin delocalization and block the internalization step of endocytosis in Saccharomyces cerevisiae.

Four mutants defective in endocytosis were isolated by screening a collection of temperature-sensitive yeast mutants. Three mutations define new END genes: end5-1, end6-1, and end7-1. The fourth mutation is in END4, a gene identified previously. The end5-1, end6-1, and end7-1 mutations do not affect vacuolar protein localization, indicating that the defect in each mutant is specific for internalization at the plasma membrane. Interestingly, localization of actin patches on the plasma membrane is affected in each of the mutants. end5-1, end6-1, and end7-1 are allelic to VRP1, RVS161, and ACT1, respectively. VRP1 and RVS161 are required for correct actin localization and ACT1 encodes actin. To our surprise, the end6-1 mutation fails to complement the act1-1 mutation. Disruption of the RVS167 gene, which is homologous to END6/RVS161 and which is also required for correct actin localization, also blocks endocytosis. The end7-1 mutant allele has a glycine 48 to aspartic acid substitution in the DNase I-binding loop of actin. We propose that Vrp1p, Rvs161p, and Rvs167p are components of a cytoskeletal structure that contains actin and fimbrin and that is required for formation of endocytic vesicles at the plasma membrane.

End4p/Sla2p interacts with actin-associated proteins for endocytosis in Saccharomyces cerevisiae.

end4-1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.

Specific sterols required for the internalization step of endocytosis in yeast.

Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Delta, erg6Delta, and erg2Deltaerg6Delta) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergDelta mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergDelta mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalization at 37 degrees C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization.

Multiple classes of yeast mutants are defective in vacuole partitioning yet target vacuole proteins correctly.

In Saccharomyces cerevisiae the vacuoles are partitioned from mother cells to daughter cells in a cell-cycle-coordinated process. The molecular basis of this event remains obscure. To date, few yeast mutants had been identified that are defective in vacuole partitioning (vac), and most such mutants are also defective in vacuole protein sorting (vps) from the Golgi to the vacuole. Both the vps mutants and previously identified non-vps vac mutants display an altered vacuolar morphology. Here, we report a new method to monitor vacuole inheritance and the isolation of six new non-vps vac mutants. They define five complementation groups (VAC8-VAC12). Unlike mutants identified previously, three of the complementation groups exhibit normal vacuolar morphology. Zygote studies revealed that these vac mutants are also defective in intervacuole communication. Although at least four pathways of protein delivery to the vacuole are known, only the Vps pathway seems to significantly overlap with vacuole partitioning. Mutants defective in both vacuole partitioning and endocytosis or vacuole partitioning and autophagy were not observed. However, one of the new vac mutants was additionally defective in direct protein transport from the cytoplasm to the vacuole.

Inhibition of Telomerase Activity Using an EGFP-Intron Splicing System Encoding Multiple RNAi Sequences.

To inhibit telomerase activity, a construct which contains artificial introns in the enhanced green fluorescent protein (EGFP) gene that encodes small hairpin RNA (shRNA) sequences that target human telomerase reverse transcriptase (hTERT) gene expression was designed and tested for its effect on lung cancer cell line. On intron splicing from the construct, intronic sequences were released and formed shRNA in the cells. After transfection of the construct, hTERT mRNA expression decreased by approximately 55 % in A549 cells. Correspondingly, in the same cell line, telomerase activity was decreased by approximately 23 %. The telomerase activity was transiently inhibited by this non-viral shRNA expression system that uses intron splicing to release artificial introns in an EGFP marker gene that contain shRNA targeting telomerase.

A continent-wide analysis of the shade requirements of red and western grey kangaroos.

Foraging time may be constrained by a suite of phenomena including weather, which can restrict a species' activity and energy intake. This is recognized as pivotal for many species whose distributions are known to correlate with climate, including kangaroos, although such impacts are rarely quantified. We explore how differences in shade seeking, a thermoregulatory behavior, of 2 closely-related kangaroo species, Macropus rufus (red kangaroos) and M. fuliginosus (western grey kangaroos), might reflect differences in their distributions across Australia. We observed foraging and shade-seeking behavior in the field and, together with local weather observations, calculated threshold radiant temperatures (based on solar and infrared radiant heat loads) over which the kangaroos retreated to shade. We apply these calculated tolerance thresholds to hourly microclimatic estimates derived from daily-gridded weather data to predict activity constraints across the Australian continent over a 10-year period. M. fuliginosus spent more time than M. rufus in the shade (7.6 ± 0.7 h versus 6.4 ± 0.9 h) and more time foraging (11.8 ± 0.5 h vs. 10.0 ± 0.6 h), although total time resting was equivalent (∼8.2 h). M. rufus tolerated 19°C higher radiant temperatures than M. fuliginosus (89°C versus 70°C radiant temperature). Across Australia, we predicted M. fuliginosus to be more restricted to shade than M. rufus, with higher absolute shade requirements farther north. These results corroborate previous findings that M. rufus is more adept at dealing with heat than M. fuliginosus and indicate that M. rufus is less dependent on shade on a continental scale.

Molecular requirements for the internalisation step of endocytosis: insights from yeast.

Molecular genetic studies of endocytosis using the unicellular eukaryote Saccharomyces cerevisiae (budding yeast) have led to the identification of many cellular components, both proteins and lipids, required for this process. While initially, many of these requirements (e.g. for actin, various actin-associated proteins, the ubiquitin conjugation system, and for ergosterol and sphingolipids) appeared to differ from known requirements for endocytosis in higher eukaryotes (e.g. clathrin, AP-2, dynamin), it now seems that endocytosis in higher and lower eukaryotes share many requirements. Often, what were initially identified as actin cytoskeleton-associated proteins in S. cerevisiae, are now revealing themselves as clathrin-coated pit- and vesicle-associated proteins in higher eukaryotes. So rather than delineating two endocytic pathways, one actin-based and one clathrin-based, the combined studies on higher and lower eukaryotes are revealing interesting interplay in both systems between the actin cytoskeleton, clathrin coats, and lipids in the formation of endocytic vesicles at the plasma membrane. Recent results from the yeast system show that the Arp2/3p complex, Wiskott-Aldrich syndrome protein (WASP), and WASP-interacting protein (WIP), proteins involved in the nucleation step of actin filament assembly, play a major role in the formation of endocytic vesicles. This discovery suggests models whereby endocytic vesicles may be actively pushed from the plasma membrane and into the cell by newly forming and rapidly extending actin filaments.

The chloroplast beta-subunit allows assembly of the Escherichia coli F0 portion of the energy transducing adenosine triphosphatase.

The effect of the expression of the chloroplast F1-ATPase beta-subunit in two Escherichia coli beta-subunit mutant strains was investigated. The amount of chloroplast beta-subunit formed in E. coli was increased by introducing a 'Shine-Dalgarno' sequence upstream from the translation start site. The chloroplast beta-subunit was membrane bound but was unable to functionally replace the mutant beta-subunit in a strain carrying the uncD409 allele [corrected]. However, in an E. coli mutant strain unable to form the beta- and epsilon-subunits the presence of the chloroplast beta-subunit enabled the assembly of a functional proton pore [corrected]