Cryo electron tomography of vitrified fibroblasts: microtubule plus ends in situ

Mouse embryonic fibroblasts (MEFs) are cells that have highly suitable biophysical properties for cellular cryo electron tomography. MEFs can be grown directly on carbon supported by EM grids. They stretch out and grow thinner than 500 nm over major parts of the cell, attaining a minimal thickness of 50 nm at their cortex. This facilitates direct cryo-fixation by plunge-freezing and high resolution cryo electron tomography. Both by direct cryo electron microscopy projection imaging and cryo electron tomography of vitrified MEFs we visualized a variety of cellular structures like ribosomes, vesicles, mitochondria, rough endoplasmatic reticulum, actin filaments, intermediate filaments and microtubules.MEFs are primary cells that closely resemble native tissue and are highly motile. Therefore, they are attractive for studying cytoskeletal elements. Here we report on structural investigations of microtubule plus ends. We were able to visualize single frayed protofilaments at the microtubule plus end in vitrified fibroblasts using cryo electron tomography. Furthermore, it appeared that MEFs contain densities inside their microtubules, although 2.5–3.5 times less than in neuronal cells [Garvalov, B.K., Zuber, B., Bouchet-Marquis, C., Kudryashev, M., Gruska, M., Beck, M., Leis, A., Frischknecht, F., Bradke, F., Baumeister, W., Dubochet, J., and Cyrklaff, M. 2006. Luminal particles within cellular microtubules. J. Cell Biol. 174, 759–765]. Projection imaging of cellular microtubule plus ends showed that 40% was frayed, which is two times more than expected when compared to microtubule growth and shrinkage rates in MEFs. This suggests that frayed ends might be stabilized in the cell cortex.     

Titin isoform switching is a major cardiac adaptive response in hibernating grizzly bears

The hibernation phenomenon captures biological as well as clinical interests to understand how organs adapt. Here we studied how hibernating grizzly bears (Ursus arctos horribilis) tolerate extremely low heart rates without developing cardiac chamber dilation. We evaluated cardiac filling function in unanesthetized grizzly bears by echocardiography during the active and hibernating period. Because both collagen and titin are involved in altering diastolic function, we investigated both in the myocardium of active and hibernating grizzly bears. Heart rates were reduced from 84 beats/min in active bears to 19 beats/min in hibernating bears. Diastolic volume, stroke volume, and left ventricular ejection fraction were not different. However, left ventricular muscle mass was significantly lower (300 +/- 12 compared with 402 +/- 14 g; P = 0.003) in the hibernating bears, and as a result the diastolic volume-to-left ventricular muscle mass ratio was significantly greater. Early ventricular filling deceleration times (106.4 +/- 14 compared with 143.2 +/- 20 ms; P = 0.002) were shorter during hibernation, suggesting increased ventricular stiffness. Restrictive pulmonary venous flow patterns supported this conclusion. Collagen type I and III comparisons did not reveal differences between the two groups of bears. In contrast, the expression of titin was altered by a significant upregulation of the stiffer N2B isoform at the expense of the more compliant N2BA isoform. The mean ratio of N2BA to N2B titin was 0.73 +/- 0.07 in the active bears and decreased to 0.42 +/- 0.03 (P = 0.006) in the hibernating bears. The upregulation of stiff N2B cardiac titin is a likely explanation for the increased ventricular stiffness that was revealed by echocardiography, and we propose that it plays a role in preventing chamber dilation in hibernating grizzly bears. Thus our work identified changes in the alternative splicing of cardiac titin as a major adaptive response in hibernating grizzly bears.     

Energy expenditure during flight in relation to body mass: effects of natural increases in mass and artificial load in Rose Coloured Starlings

Rose Coloured Starlings (Sturnus roseus) flew repeatedly for several hours in a wind tunnel while undergoing spontaneous variation in body mass. The treatments were as follows: flying unrestrained (U), with a control harness of 1.2% of their body mass (C), or with a harness of 7.4% of their body mass, which was either applied immediately before the flight (LS) or at least 9 days in advance (LL). Energy expenditure during flight (ef in W) was measured with the Doubly Labelled Water method. Flight costs in L(S) and LL were not significantly different and therefore were pooled (L). The harness itself did not affect ef, i.e. U and C flights were not different. ef was allometrically related with body mass m (in g). The slopes were not significantly different between the treatments, but ef was increased by 5.4% in L compared to C flights (log10(ef) = 0.050 + 0.47 x log10(m) for C, and log10(ef) = 0.073 + 0.47 x log10(m) for L). The difference in ef between C, LS and LL was best explained by taking the transported mass m transp (in g) instead of m into account (log10(ef) = -0.08 + 0.54 x log10(m transp)). Flight costs increased to a lesser extent than expected from interspecific allometric comparison or aerodynamic theory, regardless of whether the increase in mass occurred naturally or artificially. We did not observe an effect of treatment on breast muscle size and wingbeat frequency. We propose that the relatively low costs at a high mass are rather a consequence of immediate adjustments in physiology and/or flight behaviour than of long-term adaptations.     

Influence of adhesion to activated carbon particles on the viability of waterborne pathogenic bacteria under flow

In rural areas around the world, people often rely on water filtration plants using activated carbon particles for safe water supply. Depending on the carbon surface, adhering microorganisms die or grow to form a biofilm. Assays to assess the efficacy of activated carbons in bacterial removal do not allow direct observation of bacterial adhesion and the determination of viability. Here we propose to use a parallel plate flow chamber with carbon particles attached to the bottom plate to study bacterial adhesion to individual carbon particles and determine the viability of adhering bacteria. Observation and enumeration is done after live/dead staining in a confocal laser scanning microscope. Escherichiae coli adhered in higher numbers than Raoultella terrigena, except to a coconut-based carbon, which showed low bacterial adhesion compared to other wood-based carbon types. After adhesion, 83-96% of the bacteria adhering to an acidic carbon were dead, while on a basic carbon 54-56% were dead. A positively charged, basic carbon yielded 76-78% bacteria dead, while on a negatively charged coconut-based carbon only 32-37% were killed upon adhesion. The possibility to determine both adhesion as well as the viability of adhering bacteria upon adhesion to carbon particles is most relevant, because if bacteria adhere but remain viable, this still puts the water treatment system at risk, as live bacteria can grow and form a biofilm that can then be shedded to cause contamination.     

Mutation in the Fas pathway impairs CD8+ T cell memory

Fas death pathway is important for lymphocyte homeostasis, but the role of Fas pathway in T cell memory development is not clear. We show that whereas the expansion and contraction of CD8+ T cell response against Listeria monocytogenes were similar for wild-type (WT) and Fas ligand (FasL) mutant mice, the majority of memory CD8+ T cells in FasL mutant mice displayed an effector memory phenotype in the long-term in comparison with the mainly central memory phenotype displayed by memory CD8+ T cells in WT mice. Memory CD8+ T cells in FasL mutant mice expressed reduced levels of IFN-gamma and displayed poor homeostatic and Ag-induced proliferation. Impairment in CD8+ T cell memory in FasL mutant hosts was not due to defective programming or the expression of mutant FasL on CD8+ T cells, but was caused by perturbed cytokine environment in FasL mutant mice. Although adoptively transferred WT memory CD8+ T cells mediated protection against L. monocytogenes in either the WT or FasL mutant hosts, FasL mutant memory CD8+ T cells failed to mediate protection even in WT hosts. Thus, in individuals with mutation in Fas pathway, impairment in the function of the memory CD8+ T cells may increase their susceptibility to recurrent/latent infections.     

Catabolism of polyamines in the rat: Polyamines and their non-α-amino acid metabolites

The metabolic fate of stable isotopically labeled polyamines was investigated after their first and second intraperitoneal injection in rats. Using gas chromatographic and mass fragmentographic analyses of acid-hydrolyzed 24-h urines, some aspects of the polyamine metabolism could be elucidated. After the injections with hexadeutero-1,3-diaminopropane, obly labeled 1,3-diaminopropane was recovered from the urine samples. The rat injected with tetradeuteroputrescine excreted labeled putrescine excreted labeled putrescine, γ-amino-n-butyric acid, 2-hydroxyputrescine and spermidine, while the urine samples of the rat after the injections with tetradeuterocadaverine contained labeled cadaverine and δ-aminovaleric acid. The injections of hexadeuterospermidine led to the appearance of labeled spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-amino-n-butyric acid, putrescine, γ-amino-n-butyric acid, 1,3-diaminopropane, β-alanine and spermine. After the injections with octadeuterospermine, labeled spermine, N-(3-aminopropyl)-N′-(2-carboxyethyl)-1,4-diaminobutane, N,N′-bis(2-carboxyethyl)-1,4-diaminobutane, spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-amino-n-butyric acid, putrescine, 1,3-diaminopropane, β-alanine, 2-hydroxyputrescine and possibly γ-amino-n-butyric acid were recovered. Clear differences between the metabolism after the first and second injection were noted for putrescine, spermidine and spermine, which is suggestive for enzyme induction and/or the existence of salvage pathways.     

MORPHOLOGY OF THE EYE AND SURROUNDING STRUCTURES OF THE BOWHEAD WHALE, BALAENA MYSTICETUS

Observations were made on eyes from 46 bowhead whales, Balaena mysticetus , taken in the subsistence harvest near Barrow, Point Hope, Savoonga, and Kaktovik, Alaska. Data reported here include palpebral, eyeball, corneal, scleral, pupillary, and lens dimensions. These quantitative data have allowed us to compare structures relative to one another and sometimes to compare them with similar structures in other species. We found, for example, that the cornea is almost three times as thick at its periphery as at its center; that when the ratio of scleral thickness and eyeball size are compared, the ratio, in the bowhead whale, is twice that of any other cetacean for which data were available; and that the corneal and pupillary width to height ratios indicate a less elongated cornea and pupil than has been reported in other cetaceans. We also found a strong correlation between body length and eyeball size indicating that within the species, unlike what is seen between species, larger animals have larger eyes. Novel observations include the presence of three periorbital fatty layers, 112 ciliary processes, the presence of scleral canals, the absence of an obvious fovea or macular region in the retina, a holangiotic pattern of fundic vessels, the presence of zonular fibers and a lens sheath, and the absence of an obvious pupillary operculum. Anatomical features like the wide angle of divergence and the palpebral dimensions suggest the absence of binocular vision while features like the size of the palpebral sac, abundant conjuctival fat, and the prominence of the retractor bulbi muscle suggest mechanisms for the protrusion and retraction of the eyeball.     

Effects of NaCl on the nitrogen metabolism of the halophyteArthrocnemum fruticosum (L.) Moq., grown in a greenhouse

Summary Arthrocnemum fruticosum (L.)Moq., a halophyte from the shore of the Dead Sea in Jordan was grown in a greenhouse with nutrient solution supplemented with various concentrations of NaCl. It was shown that with increasing salinity the plants became more succulent, mainly due to an accumulation of sodium and water. Sodium was taken up into the roots in equal amounts to chloride, but in the shoots far more sodium than chloride was found, suggesting a control of these ions either in the excretion into the xylem, or in the uptake by the shoot out of the xylem. Ammonium and nitrate in the plants decreased with time on nutrient solution more or less independently of the salt concentration. However, more nitrate appeared again in the plants when they started flowering. After an initial period of adaptation the nitrate reductase activityin vivo was not inhibited by a salinity of up to 2%, but at higher NaCl concentrations a shift of nitrate reductase activity occurred from the roots to the shoots. This was consistent with earlier observations in the field. In the vegetative phase of the plants the nitrate reductase in the roots was influenced by the soil water potential, but in the shoot it was mainly dependent on the supply of nitrate from the roots. High NaCl concentrations delayed flower initiation. During flowering the nitrate reductase was involved in the re-allocation of nitrogenous compounds from the roots to the developing flowers, and it became effectively independent from salinity.     

Method for Collecting Air-Water Interface Microbes Suitable for Subsequent Microscopy and Molecular Analysis in both Research and Teaching Laboratories

A method has been developed for collecting air-water interface (AWI) microbes and biofilms that enables analysis of the same sample with various combinations of bright-field and fluorescence light microscopy optics, scanning and transmission electron microscopy (TEM), and atomic force microscopy. The identical sample is then subjected to molecular analysis. The sampling tool consists of a microscope slide supporting appropriate substrates, TEM grids, for example, that are removable for the desired protocols. The slide with its substrates is then coated with a collodion polymer membrane to which in situ AWI organisms adhere upon contact. This sampling device effectively separates the captured AWI bacterial community from the bulk water community immediately subtending. Preliminary data indicate that the AWI community differs significantly from the water column community from the same sample site when both are evaluated with microscopy and with 16S ribosomal DNA sequence-based culture-independent comparisons. This microbe collection method can be used at many levels in research and teaching.