Variable subcellular localization of glycosphingolipids

Although most glycosphingolipids (GSLs) are thought to be locatedin the outer leaflet of the plasma membrane, recent evidenceindicates that GSLs are also associated with intracellular organelles.We now report that the subcellular localization of GSLs variesdepending on the GSL structure and cell type. GSL localizationwas determined by indirect immunofluorescence microscopy offixed permeabilized cells. A single GSL exhibited variable subcellularlocalization in different cells. For example, antibody to GalCeris localized primarily to the plasma membrane of HaCaT II-3keratinocytes, but to intracellular organelies in other epithelialcells. GalCer is localized to small vesicles and tubulovesicularstructures in MDCK cells, and to the surface of phase-denselipid droplets in HepG2 hepatoma cells. Furthermore, withina single cell type, individual GSLs were found to exhibit differentpatterns of subcellular localization. In HepG2 cells, LacCerwas associated with small vesicles, which differed from thephase-dense vesicles stained by anti-GalCer, and Gb4Cer wasassociated with the intermediate filaments of the cytoskeleton.Both anti-GalCer and monoclonal antibody A2B5, which binds polysialogangliosides,localized to mitochondria. The distinct subcellular localizationpatterns of GSLs raise interesting questions about their functionsin different organelles. Together with published data on theenrichment of GSLs in specific organelles and in apical plasmamembrane, these findings indicate the existence of specificsorting mechanisms that regulate the intracellular transportand localization of GSLs. cytoskeleton glycosphingolipid intracellular organelles mitochondria subcellular localization     

The challenge of estimating kelp production in a turbid marine environment

Coastal kelp forests produce substantial marine carbon due to high annual net primary production (NPP) rates, but upscaling of NPP estimates over time and space remains difficult. We investigated the impact of variable underwater photosynthetically active radiation (PAR) and photosynthetic parameters on photosynthetic oxygen production of Laminaria hyperborea, the dominant NE-Atlantic kelp species, throughout summer 2014. Collection depth of kelp had no effect on chlorophyll a content, pointing to a high photoacclimation potential of L. hyperborea towards incident light. However, chlorophyll a and photosynthesis versus irradiance parameters differed significantly along the blade gradient when normalized to fresh mass, potentially introducing large uncertainties in NPP upscaling to whole thalli. Therefore, we recommend a normalization to kelp tissue area, which is stable over the blade gradient. Continuous PAR measurements revealed a highly variable underwater light climate at our study site (Helgoland, North Sea) in summer 2014, reflected by PAR attenuation coefficients (K_d) between 0.28 and 0.87 m⁻¹. Our data highlight the importance of continuous underwater light measurements or representative average values using a weighted K_d to account for large PAR variability in NPP calculations. Strong winds in August increased turbidity, resulting in a negative carbon balance at depths >3–4 m over several weeks, considerably impacting kelp productivity. Estimated daily summer NPP over all four depths was 1.48 ± 0.97 g C · m⁻² seafloor · d⁻¹ for the Helgolandic kelp forest, which is in the range of other kelp forests along European coastlines.     

REPRODUCTIVE ATTRITION IN THE RARE CHAPARRAL SHRUB FREMONTODENDRON DECUMBENS LLOYD (STERCULIACEAE)

We examined reproductive attrition in Fremontodendron decumbens to characterize sexual reproduction in this rare California shrub. Reproductive individuals produced an average of 2,900 flower buds in a season, with no significant difference in bud production between two seasons. Because of intense insect predation, <;2% of initiated flower buds became mature fruits. A threefold decrease in predation of flower buds between seasons resulted in an increase in seed output the second season, indicating that seed production was partially predator-limited. Most seeds (97.8%) were dormant due to an impermeable seed coat. Breaking of the coat, mechanically or by heat, allowed high levels of germination. Chamise charate and ash added to the potting medium resulted in the highest level of germination and emergence. Rodents were more important than birds as seed predators, destroying 90% of seeds under parent shrub canopies within 8–10 months. Seeds already integrated into the seed bank were comparatively safe from predation, relative to newly added seeds. If predation was prevented, seeds were long-lived under field conditions (>;80% survived after 5.75 years). Most seedlings produced in unburned chaparral by planting heat-treated seeds in openings between shrubs were destroyed by predators (rodents and insects). All seedlings that escaped predation died during the summer drought. We concluded that sexual reproduction was limited by (in order of importance): 1) lack of fire, 2) predehiscence predation by insects, and 3) postdehiscence predation by rodents. Size distributions from two populations revealed that, despite the apparent absence of sexual reproduction in unbumed chaparral, two unbumed sites contained a large proportion of individuals in small size classes. Excavation of several small individuals demonstrated they were sprouts from the roots of nearby larger shrubs. Because asexual reproduction by rootsprouting circumvents the high attrition of sexual reproductive effort on unbumed sites, rootsprouting may be a significant reproductive strategy of some ‘sprouter’ species in chaparral.     

Development of a new method for obtaining osteoclasts from endosteal surfaces

Summary Techniques for the isolation of ahhighly pure population of viable osteoclasts are limited. For this reason, we developed an isolation procedure that results in a high yield of osteoclast-like cells, up to 92% pure, from 3-wk-old chicken tibias. The unique feature of the method is the migration of cells from marrow-free endosteal surfaces to vitronectin-coated plates. The cells retain the osteoclast phenotype and remain viable in culture for a minimum of 1 wk. The cells were characterized and compared to two populations of authentic avian osteoclasts, which were isolated on the basis of association with fibronectin-coated plates. The cells contained substantial amounts of tartrate-resistant acid phosphatase. Alkaline phosphatase levels were negligible, suggesting little contamination by osteoblasts. Response to parathyroid hormone, dibutyryl cyclic adenosine monophosphate, calcitonin, acetazolamide, 17β-estradiol, and prostaglandin E₂ was evident, as detected by measuring acid production. The vitronectin-associating cells contained numerous mitochondria, had the ability to resorb bone in anin vitro bone slice assay, and specifically bound biotinylated vitronectin. At 5 d of culture, the cells demonstrated marginal multinuclearity, having two to three nuclei. A large number (∼1×10⁶ cells/tibia) of viable cells that exhibit characteristics of authentic osteoclasts can be obtained by the method described. Potentially, this method could be applied to other species.     

Sperm motility in the presence of boundaries

The fluid dynamics of sperm motility near both rigid and elastic walls is studied using the immersed boundary method. Simulations of both single and interacting organisms are presented. In particular, we find that nearby organisms originally undulating with a 90° phase shift may adjust their relative swimming velocities and phase-lock. Comparisons with previous analytical results are also discussed. The tendency of a near-wall to attract organisms is demonstrated.     

Factors involved in capillary growth in the heart

Growth of capillaries in the heart occurs under physiological circumstances during endurance exercise training, exposure to high altitude and/or cold, and changes in cardiac metabolism or heart rate elicited by modification of thyroid hormone levels. Capillary growth in all these conditions can be linked with increased coronary blood flow, decreased heart rate, or both. This paper brings evidence that, although increased blood flow due to long-term administration of coronary vasodilators results in capillary growth, a long-term decrease in heart rate induced by electrical bradycardial pacing in rabbits and pigs, or by chronic administration of a bradycardic drug, alinidine, in rats, stimulates capillary growth with little or no change in coronary blood flow. Decreased heart rate results in increased capillary wall tension, increased end-diastolic volume and increased force of contraction, and thus stretch of the capillary wall. This could lead to release of various growth factors possibly stored in the capillary basement membrane. Correlation was found between capillary density (CD) and the levels of low molecular endothelial cell stimulating angiogenic factor (ESAF) both in rabbit and pig hearts with CD increased by pacing. There was no relation between expression of mRNA for basic fibroblast growth factor and CD in sham-operated and paced rabbit hearts. In contrast, mRNA for TGFß was increased in paced hearts, and the possible role of this factor in the regulation of capillary growth induced by bradycardia is discussed.     

Domain-swapping analysis of FtsI, FtsL, and FtsQ, bitopic membrane proteins essential for cell division in Escherichia coli.

FtsI, FtsL, and FtsQ are three membrane proteins required for assembly of the division septum in the bacterium Escherichia coli. Cells lacking any of these three proteins form long, aseptate filaments that eventually lyse. FtsI, FtsL, and FtsQ are not homologous but have similar overall structures: a small cytoplasmic domain, a single membrane-spanning segment (MSS), and a large periplasmic domain that probably encodes the primary functional activities of these proteins. The periplasmic domain of FtsI catalyzes transpeptidation and is involved in the synthesis of septal peptidoglycan. The precise functions of FtsL and FtsQ are not known. To ask whether the cytoplasmic domain and MSS of each protein serve only as a membrane anchor or have instead a more sophisticated function, we have used molecular genetic techniques to swap these domains among the three Fts proteins and one membrane protein not involved in cell division, MalF. In the cases of FtsI and FtsL, replacement of the cytoplasmic domain and/or MSS resulted in the loss of the ability to support cell division. For FtsQ, MSS swaps supported cell division but cytoplasmic domain swaps did not. We discuss several potential interpretations of these results, including that the essential domains of FtsI, FtsL, and FtsQ have a role in regulating the localization and/or activity of these proteins to ensure that septum formation occurs at the right place in the cell and at the right time during the division cycle.     

Respiratory function of velopharyngeal constrictor muscles during wakefulness in normal adults

Launois, Sandrine H., Judy Tsui, and J. Woodrow Weiss.Respiratory function of velopharyngeal constrictor muscles during wakefulness in normal adults. J. Appl.Physiol. 82(2): 584-591, 1997.The levator velipalatini (LVP) and the superior pharyngeal constrictor (SPC) influencevelopharyngeal patency and soft palate position, but their behaviorduring respiration is incompletely characterized. To further clarifytheir respiratory function, we recorded electromyographic activity(EMG) in the LVP and the SPC in awake normal subjects breathing orally.EMG data were obtained in six subjects for the LVP and in nine subjectsfor the SPC. EMG activity and timing and ventilation were measuredduring isocapnic hypoxia and hyperoxic hypercapnia. Phasic EMG activitywas inconsistently present during unstimulated oral breathing. Timingof EMG phasic activity was variable for both muscles. Peak LVP activitywas mainly or exclusively expiratory in three of six subjects. Peak SPCactivity was mainly or exclusively expiratory in five of nine subjects.With chemostimulation, recruitment of phasic activity was observed inthe LVP in four of six subjects and in the SPC in five of ninesubjects. Tonic activity increased in four of six subjects for the LVPand in three of nine subjects for the SPC. However, the response wasalinear, and intersubject as well as breath-to-breath variability wassubstantial. In conclusion, LVP and SPC are characterized by the highinter- and intrasubject variability of EMG activity, timing ofactivation, and response to chemostimulation.

     

Postfertilization causes of differential success of pollen donors in Erythronium grandiflorum (Liliaceae): nonrandom ovule abortion

Seed paternity in Erythronium grandiflorum does not fully reflect the proportion of pollen on the stigma. When two types of pollen are simultaneously applied to the stigma, outbred seeds are produced over inbred, and seeds from more distant donors are produced over seeds from donors nearby. I looked for postfertilization causes of these previously reported patterns of differential success of pollen donors. I simultaneously pollinated stigmas with pollen from two donors and observed ovule development through a window sliced in the ovary. Pollen donor pairs were self and cross, donors 1 and 100 m from the recipient, and two donors each 100 m from the recipient. Since one donor was always the alternate homozygote from the recipient at the malate dehydrogenase locus, I could determine the paternity of developing seeds. When it appeared that ovules were aborting, I removed them and determined their paternity using starch gel electrophoresis. Ovules fertilized by self pollen were more likely to abort than ovules fertilized by cross pollen, and ovules fertilized by nearby donors were more likely to abort than ovules fertilized by distant donors. Ovules fertilized by donors 100 m from the recipient were equally likely to abort. There was not a significant relationship between the proportion of ovules fertilized by a pollen donor and the probability of those fertilized ovules developing into seeds. There was no relationship between ovule position within a fruit and ovule abortion. I manipulated available resources by removing leaves and by permitting only one fruit to develop per plant. Decreasing the amount of resources increased the proportion of aborted ovules. Abortion of ovules of lesser quality appears to release resources that can then be used to develop other offspring.     

Evidence for a distinct region causing a cat-like cry in patients with 5p deletions.

The cri-du-chat syndrome is a contiguous gene syndrome that results from a deletion of the short arm of chromosome 5 (5p). Patients present with a cat-like cry at birth, which is usually considered diagnostic of this syndrome. Additional features of the syndrome include failure to thrive, microcephaly, hypertelorism, epicanthal folds, hypotonia, and severe mental retardation. We report on four families in which patients with 5p deletions have only the characteristic cat-like cry, with normal to mildly delayed development. The precise locations of the deletions in each family were determined by FISH using lambda phage and cosmid clones. All of the deletion breakpoints map distal to a chromosomal region that is implicated with the facial features and severe mental and developmental delay in the cri-du-chat syndrome. DNA clones mapping in the chromosomal region associated with the cat-like cry feature will be useful diagnostic tools. They will allow for the distinction between 5p deletions that will result in the severe delay observed in most cri-du-chat syndrome patients and those deletions that result in the isolated cat-like cry feature, which is associated with a better prognosis.