Biophysics of zebrafish (Danio rerio) sperm

In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37 ± 0.02 (SEM), an isosmotic cell volume (V_o) of 12.1 ± 0.2 μm³ (SEM), a water permeability (L_p) in 10% dimethyl sulfoxide of 0.021 ± 0.001(SEM) μm/min/atm, and a cryoprotectant permeability (P_s) of 0.10 ± 0.01 (SEM) × 10⁻³ cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24 h at 0 °C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.     

Correlating Global Gene Regulation to Angiogenesis in the Developing Chick Extra-Embryonic Vascular System

Background

Formation of blood vessels requires the concerted regulation of an unknown number of genes in a spatial-, time- and dosage-dependent manner. Determining genes, which drive vascular maturation is crucial for the identification of new therapeutic targets against pathological angiogenesis.

Methology/Principal Findings

We accessed global gene regulation throughout maturation of the chick chorio-allantoic membrane (CAM), a highly vascularized tissue, using pan genomic microarrays. Seven percent of analyzed genes showed a significant change in expression (>2-fold, FDR<5%) with a peak occurring from E7 to E10, when key morphogenetic and angiogenic genes such as BMP4, SMO, HOXA3, EPAS1 and FGFR2 were upregulated, reflecting the state of an activated endothelium. At later stages, a general decrease in gene expression occurs, including genes encoding mitotic factors or angiogenic mediators such as CYR61, EPAS1, MDK and MYC. We identified putative human orthologs for 77% of significantly regulated genes and determined endothelial cell enrichment for 20% of the orthologs in silico. Vascular expression of several genes including ENC1, FSTL1, JAM2, LDB2, LIMS1, PARVB, PDE3A, PRCP, PTRF and ST6GAL1 was demonstrated by in situ hybridization. Up to 9% of the CAM genes were also overexpressed in human organs with related functions, such as placenta and lung or the thyroid. 21–66% of CAM genes enriched in endothelial cells were deregulated in several human cancer types (P<.0001). Interfering with PARVB (encoding parvin, beta) function profoundly changed human endothelial cell shape, motility and tubulogenesis, suggesting an important role of this gene in the angiogenic process.

Conclusions/Significance

Our study underlines the complexity of gene regulation in a highly vascularized organ during development. We identified a restricted number of novel genes enriched in the endothelium of different species and tissues, which may play crucial roles in normal and pathological angiogenesis.     

Structure and inhibitory effects on angiogenesis and tumor development of a new vascular endothelial growth inhibitor

Blocking angiogenesis is an attractive strategy to inhibit tumor growth, invasion, and metastasis. We describe here the structure and the biological action of a new cyclic peptide derived from vascular endothelial growth factor (VEGF). This 17-amino acid molecule designated cyclopeptidic vascular endothelial growth inhibitor (cyclo-VEGI, CBO-P11) encompasses residues 79-93 of VEGF which are involved in the interaction with VEGF receptor-2. In aqueous solution, cyclo-VEGI presents a propensity to adopt a helix conformation that was largely unexpected because only beta-sheet structures or random coil conformations have been observed for macrocyclic peptides. Cyclo-VEGI inhibits binding of iodinated VEGF165 to endothelial cells, endothelial cells proliferation, migration, and signaling induced by VEGF165. This peptide also exhibits anti-angiogenic activity in vivo on the differentiated chicken chorioallantoic membrane. Furthermore, cyclo-VEGI significantly blocks the growth of established intracranial glioma in nude and syngeneic mice and improves survival without side effects. Taken together, these results suggest that cyclo-VEGI is an attractive candidate for the development of novel angiogenesis inhibitor molecules useful for the treatment of cancer and other angiogenesis-related diseases.     

Leucokinin and diuretic hormone immunoreactivity of neurons in the tobacco hornworm,Manduca sexta,and co-localization of this immunoreactivity in lateral neurosecretory cells of abdominal ganglia

Because leucokinins stimulate diuresis in some insects, we wished to identify the neurosecretory cells in Manduca sexta that might be a source of leucokinin-like neurohormones. Immunostaining was done at various stages of development, using an antiserum to leucokinin IV. Bilateral pairs of neurosecretory cells in abdominal ganglia 3–7 of larvae and adults are immunoreactive; these cells project via the ipsilateral ventral nerves to the neurohemal transverse nerves. The immunoreactivity and size of these lateral cells greatly increases in the pharate adult, and this change appears to be related to a period of intensive diuresis occurring a few days before adult eclosion. Relationships of these neurons to cells that are immunoreactive to a M. sexta diuretic hormone were also investigated. Diuretic hormone and leucokinin immunoreactivity are co-localized in the lateral neurosecretory cells and their neurohemal projections. A median pair of leucokinin-immunoreactive, and a lateral pair of diuretic hormone-immunoreactive neurons in the larval terminal abdominal ganglion project to neurohemal release sites within the cryptonephridium. The immunoreactivity of these cells is lost as the cryptonephridium is eliminated during metamorphosis. This loss appears to be related to the change from the larval to adult pattern of diuresis.     

Untersuchungen über das Transportsystem für Thiamin bei Bacillus cereus

Zusammenfassung Bei dem untersuchten Transportsystem für Thiamin bei Bacillus cereus wechseln rhythmisch 1–2 h andauernde Aufnahme-und Abgabephasen miteinander ab. Diese großen Phasen sind in kleinere von durchschnittlich 45 s Dauer unterteilt. Die Geschwindigkeit der Thiaminaufnahme wird von pH-Wert, Temperatur, Alter der Zellen, Energie-und Nährstoffversorgung sowie Thiaminkonzentration des Mediums beeinflußt. Sie folgt der Michaelis-Menten-Kinetik: K _m =1,98x10^-8 M; V _max=1,19x10^-6 mol/g TGxmin. Gefördert wird die Aufnahmerate durch K⁺, Ca²⁺ und Mg²⁺, gehemmt wird sie durch Pyrithiamin, EDTA, H⁺-Ionen, Wasserstoffacceptoren und-donatoren; OH^--Ionen bewirken eine Umkehr der Transportrichtung. Als erklärende Theorie wird eine Kopplung der Thiaminpermeation mit Protonenverschiebungen in der Membran diskutiert.

---

Studies on the thiamine transport system in Bacillus cereus

The thiamine transport system in Bacillus cereus exhibits rhythmical changes of resorption-and excretion-phases lasting 1–2 h. These main phases are subdivided in shorter ones with an average duration of 45 s. The velocity of the thiamine uptake is influenced by pH, temperature, age of cells, energy and substrate supply and thiamine concentration of the medium. The Michaelis-Menten-Kinetic can be used to describe the uptake: K _m =1.98x10^-8 M; V _max=1.19x10^-6 mol/g dry weightxmin. The rate is enhanced by K⁺, Ca²⁺ and Mg²⁺, and inhibited by Pyrithiamin, EDTA, H⁺-ions, proton donors and proton acceptors; OH^--ions cause a change in the direction of transport. A theoretical explanation can be given by assuming a coupling of the thiamine permeation with proton movements in the membrane.

---

Liste der Abkürzungen AMP Adenosinmonophosphat - ADP Adenosindiphosphat - ATP Adenosintriphosphat - DNP Dinitrophenol - EDTA Athylendiamintetraessigsäure - FAD Flavinadenindinucleotid - NAD Nicotinamidadenindinucleotid - NADP Nicotinamidadenindinucleotidphosphat - TPP Thiaminpyrophosphat     

Influences of soil acidity on Streptomyces populations inhabiting forest soils.

The Streptomyces populations inhabiting five acidic forest soils were examined. It was found that lowering the pH of a medium selective for streptomycetes (starch-casein agar) to the pH of the particular soil horizon being plated influenced both the total numbers and types of streptomycetes that were isolated from the soils examined in this study. On the acidified medium both the numbers of streptomycetes and the percentage of total bacteria on the plates represented by streptomycetes increased (as compared with the same medium with a pH of 7.2). These differences were greatest on the isolations from the most acid soils. The largest concentrations of streptomycetes were found in the surface horizon (0 to 15 cm) and the litter layer immediately over the surface mineral horizon. Acidity tolerance tests demonstrated that random samplings of isolates contained acidophilic, neutrophilic, and acidoduric strains, with the largest numbers of acidophiles being found on the acidified media from the most acid soils. There were no differences between overall utilization of selected carbohydrates among the isolates taken from either the neutral or acidic media, although a larger proportion of the acid media isolates produced acid from the carbohydrates. Evidence is presented which indicates that different types of streptomycetes were isolated on the acid media, and possible reasons for the presence of these acid-tolerant populations are discussed.     

Treeline advances along the Urals mountain range – driven by improved winter conditions?

High‐altitude treelines are temperature‐limited vegetation boundaries, but little quantitative evidence exists about the impact of climate change on treelines in untouched areas of Russia. Here, we estimated how forest‐tundra ecotones have changed during the last century along the Ural mountains. In the South, North, Sub‐Polar, and Polar Urals, we compared 450 historical and recent photographs and determined the ages of 11 100 trees along 16 altitudinal gradients. In these four regions, boundaries of open and closed forests (crown covers above 20% and 40%) expanded upwards by 4 to 8 m in altitude per decade. Results strongly suggest that snow was an important driver for these forest advances: (i) Winter precipitation has increased substantially throughout the Urals (~7 mm decade^?1), which corresponds to almost a doubling in the Polar Urals, while summer temperatures have only changed slightly (~0.05 °C decade^?1). (ii) There was a positive correlation between canopy cover, snow height and soil temperatures, suggesting that an increasing canopy cover promotes snow accumulation and, hence, a more favorable microclimate. (iii) Tree age analysis showed that forest expansion mainly began around the year 1900 on concave wind‐sheltered slopes with thick snow covers, while it started in the 1950s and 1970s on slopes with shallower snow covers. (iv) During the 20th century, dominant growth forms of trees have changed from multistemmed trees, resulting from harsh winter conditions, to single‐stemmed trees. While 87%, 31%, and 93% of stems appearing before 1950 were from multistemmed trees in the South, North and Polar Urals, more than 95% of the younger trees had a single stem. Currently, there is a high density of seedlings and saplings in the forest‐tundra ecotone, indicating that forest expansion is ongoing and that alpine tundra vegetation will disappear from most mountains of the South and North Urals where treeline is already close to the highest peaks.     

Anthropological and palaeopathological analysis of the human remains from three "Tombs of the Nobles" of the necropolis of Thebes-west, upper Egypt

During several recent excavation campaigns at the necropolis of Sheikh-Abd-el-Gurna, Thebes-West, Upper Egypt, we investigated the human remains of three "Tombs of the Nobles" totalling at least 273 individuals. The investigation covered the human material (skeletons and mummy residues) from the tombs TT-84, TT-85 and TT-95. These tombs had been built in the New Kingdom (approx. 1500-1000 B.C.) and used until the Late period (up to 330 BC). All samples were analyzed macroscopically, isolated findings were further investigated by endoscopic and radiological techniques. The at least 273 individuals covered an age range from newborns to senile individuals with a main age of death between 20 and 40 years of age. The rate of infants and subadults was at 20.2% of all individuals and there was a slight male predominance comprising 54.5% of the adults. In this population a fairly high rate of pathological lesions was seen. Thus, dental conditions generally were poor with a high degree of dental abrasion, an also high rate of carious dental lesions (affecting between 13.8% and 27.7% of the yaws) and consequently a significant number of dental abscesses (mean 15.9%). Residues of trauma were observed in a considerable number of individuals ranging between 12.3% and 22.6% depending on the burial place (mean 15.8%). Inflammatory bone reactions (except the dental abscesses) were present to variable extent, in some locations ranging up to 6.8% of the cases (mean 5.1%). In addition, we noted several cases showing cribra orbitalia (mean 29.2%) and porotic hyperostosis (15.4% of cases), mild to severe osteopenia (7.5%) and in several cases subperiosteal new bone formation suggestive of chronic vitamin D-deficiency ("scurvy") (9.5%). The data support the notion of a significant impairment of living conditions in a high number of individuals. The rates of osteoarthrotic joint alterations were considerably variable depending on the burial places (between 1.9% and 18.5%) providing insight into the physical activities of subpopulations in different locations of the burial places. Similarly, the rate of spondylosis showed also variable extent ranging between 12% and 66.1% of vertebral bodies. In general, our analysis provides evidences that the living conditions in the ancient Egyptian population of one of the main necropoles of Thebes analyzed in this study were quite poor. This is somewhat in contrast to the usually assumed high standard of living of the socially higher population of the capital of ancient Egypt.