Structure of the human telomere in Na+ solution: an antiparallel (2+2) G-quadruplex scaffold reveals additional diversity

Single-stranded DNA overhangs at the ends of human telomeric repeats are capable of adopting four-stranded G-quadruplex structures, which could serve as potential anticancer targets. Out of the five reported intramolecular human telomeric G-quadruplex structures, four were formed in the presence of K⁺ ions and only one in the presence of Na⁺ ions, leading often to a perception that this structural polymorphism occurs exclusively in the presence of K⁺ but not Na⁺. Here we present the structure of a new antiparallel (2+2) G-quadruplex formed by a derivative of a 27-nt human telomeric sequence in Na⁺ solution, which comprises a novel core arrangement distinct from the known topologies. This structure complements the previously elucidated basket-type human telomeric G-quadruplex to serve as reference structures in Na⁺-containing environment. These structures, together with the coexistence of other conformations in Na⁺ solution as observed by nuclear magnetic resonance spectroscopy, establish the polymorphic nature of human telomeric repeats beyond the influence of K⁺ ions.     

Organization of interphase microtubules in fission yeast analyzed by electron tomography

Polarized cells, such as neuronal, epithelial, and fungal cells, all display a specialized organization of their microtubules (MTs). The interphase MT cytoskeleton of the rod-shaped fission yeast, Schizosaccharomyces pombe, has been extensively described by fluorescence microscopy. Here, we describe a large-scale, electron tomography investigation of S. pombe, including a 3D reconstruction of a complete eukaryotic cell volume at sufficient resolution to show both how many MTs there are in a bundle and their detailed architecture. Most cytoplasmic MTs are open at one end and capped at the other, providing evidence about their polarity. Electron-dense bridges between the MTs themselves and between MTs and the nuclear envelope were frequently observed. Finally, we have investigated structure/function relationships between MTs and both mitochondria and vesicles. Our analysis shows that electron tomography of well-preserved cells is ideally suited for describing fine ultrastructural details that were not visible with previous techniques.     

Calcium mediates the light-induced decrease in maintained K+ current in Limulus ventral photoreceptors

In addition to increasing the conductance to sodium, light reduces the maintained voltage-dependent potassium current (iK) in Limulus ventral photoreceptors. We have investigated the mechanism underlying this long-lasting decrease in ik. Intracellular injection of calcium produced a similar reduction of the voltage-dependent outward current. This reduction was not due to an activation of the voltage-dependent inward current (iin) because calcium injection reduced the outward current even under conditions where iin was blocked with Ni2+, and because calcium injection produced a decrease in conductance, as measured from the slope of the instantaneous i-V curve. The effect of light on ik could be blocked by injection of the calcium buffer EGTA (pCa 7.1) to an intracellular concentration of 50-70 mM. Even larger injections of the pH buffer MOPS (100-200 mM) did not reduce the effect of light on ik. These experiments show that intracellular free calcium (Cai2+) can reduce ik. Furthermore, since Cai2+ is known to increase in light, our results are consistent with the hypothesis that calcium is the internal transmitter for the light-induced decrease in ik.     

Light-regulated and organ-specific expression of types 1, 2, and 3 light-harvesting complex b mRNAs in Ginkgo biloba.

In a prior study (E. Chinn and J. Silverthorne [1993] Plant Physiol 103: 727-732) we showed that the gymnosperm Ginkgo biloba was completely dependent on light for chlorophyll synthesis and chloroplast development and that expression of light-harvesting complex b (Lhcb) mRNAs was substantially increased by light. However, dark-grown seedlings that were transferred to constant white light took significantly longer than angiosperm seedlings to initiate a program of photomorphogenesis and the stems failed to green completely. We have prepared type-specific probes for mRNAs encoding major polypeptides of light-harvesting complex II (Lhcb1, Lhcb2, and Lhcb3) and have used these to analyze the expression of individual Lhcb mRNAs during greening. All three sequences accumulated in the top portions of dark-grown seedlings transferred to light, but, as was seen previously for total Lhcb mRNAs, there was a transient, reproducible decline in the levels of all three mRNAs after 4 d in the light. This transient decrease in Lhcb mRNA levels was not paralleled by a decrease in Chl accumulation. By contrast, there were significantly lower levels of all three Lhcb mRNAs in the lower portions of greening dark-grown stems as well as lower Chl levels. We conclude that although the tops of the plants have the capacity to etiolate and green, Gingko seedling stems continue a program of development into woody tissue in darkness that precludes greening when the seedlings are transferred to the light.     

Congenital heart disease among spontaneous abortuses and stillborn fetuses: prevalence and associations

The prevalence, range, and associations of congenital heart disease (CHD) were studied among 400 spontaneous abortuses between 9 and 40 weeks' gestation. Fifty-two (13.0%) cases of CHD were detected. To minimize selection bias the specimens were grouped by external appearance and the prevalence expressed accordingly. CHD was detected in 21 (7.3%) of 289 externally normal and 31 (27.9%) of 111 externally abnormal fetuses. Ventricular septal defect (VSD) was the most frequent CHD found in isolation as well as in combination with extracardiac malformations. Seventy-five percent of isolated CHD was VSD. Forty (69.2%) of the 52 cases of CHD were associated with extracardiac malformations. Chromosomal syndromes were responsible for a minimum of 19.2% of the cases and suspected in up to 36.5%. The most frequent associations involved the musculoskeletal system, central nervous system, abdominal wall, and kidneys. In contrast, studies of liveborn infants have reported 70% of CHD as isolated defects, including many CHD infrequently seen among spontaneous abortuses. This suggests that fetuses with isolated CHD often survive to term, and CHD does not significantly affect the survival of the fetus in utero. Ventricular septum formation may be particularly susceptible to hemodynamic changes and may be indicative of an underlying pathologic condition that also leads to a spontaneous abortion.     

Production by Hexagenia limbata in a warm-water reservoir and its association with chlorophyll content of the water column

We investigated the productivity of nymphs of the mayfly Hexagenia limbata in Lake Waco, a central Texas reservoir, and assessed its association with chlorophyll content of the water. We hypothesized that food availability measured as chlorophyll content of the water may directly associate with growth of Hexagenia and predict population productivity. To test this, we compared production by mayfly populations at two stations in the same reservoir; a northern station receiving water input with high chlorophyll content, and a southern station receiving water with low chlorophyll content. Both stations had similar substrate type and abundant mayflies. Benthic samples were collected from October 1984 through September 1985, and dissolved oxygen and temperature of the water were monitored.Annual production (size-frequency method) was 1270 mg m^–2 (P/B = 7.5) at the northern station and 1990 mg m^–2 (P/B = 6.1) at the southern station. The mean standing crop was 323 mg m ^{– 2} at the southern station and 169mg m^–2 at the southern station. Densities of mayflies at the two stations were not significantly different.Mean chlorophyll concentration (total mg pigment) during the sampling period was 23.5 mg m^–3 at the northern station and 16.7 mg m ^{– 3} at the southern station. Therefore, the station with lower mean chlorophyll content had higher secondary productivity by Hexagenia. Conversely, the station with higher mean chlorophyll content had lower mayfly productivity. The productivity of the mayfly populations did not positively associate with the chlorophyll content of the water, and chlorophyll content did not predict the success of the population of Hexagenia. Variation in mayfly growth success was associated with differences in temperature and dissolved oxygen. The northern station with higher chlorophyll content and lower productivity had low dissolved oxygen and temperatures higher than optimum for growth.     

Effect of scaffold material, construct length and mechanical stimulation on the in vitro stiffness of the engineered tendon construct

Introducing mesenchymal stem cell (MSC)-seeded collagen constructs into load-protected wound sites in the rabbit patellar and Achilles tendons significantly improves their repair outcome compared to natural healing of the unfilled defect. However, these constructs would not be acceptable alternatives for repairing complete tendon ruptures because they lack the initial stiffness at the time of surgery to resist the expected peak in vivo forces thereafter. Since the stiffness of these constructs has also been shown to positively correlate with the stiffness of the subsequent repairs, improving initial stiffness by appropriate selection of in vitro culture conditions would seem crucial. In this study we examined the individual and combined effects of collagen scaffold type, construct length, and mechanical stimulation on in vitro implant stiffness. Two levels each of scaffold material (collagen gel vs. collagen sponge), construct length (short vs. long), and mechanical stimulation (stimulated vs. non-stimulated) were examined. Our results indicate that all three treatment factors influenced construct linear stiffness. Increasing the length of the construct had the greatest effect on the stiffness compared to introducing mechanical stimulation or changing the scaffold material. A significant interaction was also found between length and stimulation. Of the eight groups studied, longer, stimulated, cell-sponge constructs showed the highest in vitro linear stiffness. We now plan in vivo studies to determine if higher stiffness constructs generate higher stiffness repairs 12 weeks after surgery and if in vitro construct stiffness continues to correlate with in vivo repair parameters like linear stiffness.     

Support for the immortal strand hypothesis: neural stem cells partition DNA asymmetrically in vitro

The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates. We investigated cosegregation in neural stem cells (NSCs). After exposure to the thymidine analogue 5-bromo-2-deoxyuridine (BrdU), which labels newly synthesized DNA, a subset of neural precursor cells were shown to retain BrdU signal. It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs. This asymmetric partitioning of DNA then was demonstrated during mitosis, and these results were further supported by real time imaging of SC clones, in which older and newly synthesized DNA templates were distributed asymmetrically after DNA synthesis. We demonstrate that NSCs are unique among precursor cells in the uneven partitioning of genetic material during cell divisions.