Transition from somatic to meiotic pairing and progressional changes of the synaptonemal complex in spermatocytes of Aedes aegypti

Aedes aegypti spermatocytes were reconstructed from electron micrographs. The species has tight somatic pairing of the chromosomes, and there are therefore no classical leptotene and zygotene stages, but rather a gradual transition from somatic pairing to meiotic pairing (= pachytene). The term prepachytene has been used for the transitory stage. The first visible sign of impending meiosis was a reorganization of the chromatin, which resulted in the formation of spaces (synaptic spaces) in the chromatin, about the width of the synaptonemal complexes (SCs). Diffuse material, possibly precursor material for the SC, was present in the spaces. Later short pieces of complex were formed throughout the nucleus. Late prepachytene, pachytene, and diplotene complexes were reconstructed. Each chromosome occupied a separate region of the nucleus. The complexes became progressively shorter from prepachytene (maximum complement length 289 m) to diplotene (175 m). The thickness of the SCs increased from prepachytene to pachytene and probably decreased again during diplotene. At the beginning of diplotene the lateral elements (LEs) separated, and the single LEs became two to three times thicker than the LEs of the SC. The centromeres were at all stages attached to the nuclear membrane, whereas the telomeres were free in the nucleoplasm during pachytene and diplotene. A heterochromatic marker was present on chromosome 1 near the sex determining locus, and a diffuse marker on chromosome 3 near the nucleolus organizer region. After breakdown of the complexes, polycomplexes were present in the nucleus.     

Separation of macromolecules in isotachophoresis systems involving single or multiple counterions

The isotachcophoresis principle provides unique opportunities for rational designs of fractionation procedures involving charged molecules. Theoretically any two charged molecules that are soluble under the experimental conditions involved can be physically separated if their electrophoretic net mobilities differ only slightly in the electrophoresis medium used. A theoretical and practical outline is presented that enables the reader to set up this fractionation system and on a rational basis develop fractionation procedures for a given set of charged macromolecules by isotachophoresis with simple and well characterized ampholytes as spacer substances. The planning of preparative experiments in this approach is based on results obtained from rapid analytical screens on a microgram scale. The report includes an appendix containing the theoretical basis for computation of buffer compositions in the isotachophoretic steady state with mono/polyvalent constituents in systems involving one or more counterions and controlled amounts of interferiong ions.     

Studies on Tyzzer's disease in rats.

An outbreak of an epidemic disease occurred in a specified-pathogen-free (SPF) breeding colony of rats. The clinical signs and the post-mortem findings were characteristic for Tyzzer's disease. The causative agent, Bacillus piliformis, was demonstrated microscopically in ileum, liver and myocardium, and transmitted to mice where its pathogenicity appeared to be similar to that of another strain isolated from mice. B. piliformis from spontaneously-infected rats was demonstrated by indirect immunofluorescence technique. By means of the same technique it was found that the fluorescence antibody titre obtained of the individual sera from spontaneously-infected mice, rats and rabbits was the same, whether the antigen employed was organisms isolated from rats or mice. By testing sera from healthy rats in 3 different colonies by use of immunofluorescence technique, antibodies were found in several sera.     

A plant serpin gene. Structure, organization and expression of the gene encoding barley protein Z4

A 3133-bp nucleotide sequence of the gene Paz1 on chromosome 4 of barley, encoding endosperm protein Z4, has been determined. The sequence includes 1079 bp 5' upstream and 523 bp 3' downstream of the coding region. The 1079-bp 5' upstream region of the gene shows little similarity to 5' regions of other sequences genes expressed in the developing cereal endosperm. The coding sequence is interrupted by one 334-bp-long intron (bases 1497-1830). The deduced amino acid sequence, which was corroborated by peptide sequences, consists of 399 amino acids and has a molecular mass of 43,128 Da. This sequence confirms protein Z4 to be a member of the serpin superfamily of proteins. The similarity with other members of the family expressed as amino acids in identical positions is in the order of 25-30% and pronounced in the carboxy-terminal half of the molecule. Sequence residues assumed to form clusters stabilizing the tertiary structure are highly conserved. Protein Z4 is synthesized in the developing endosperm without a signal peptide and protein Z4 mRNA was evenly distributed among the free and membrane-bound polyribosomes of the endosperm cell. An internal hydrophobic region of 21 amino acids (residues 36-56) may serve as a signal for targeting the polypeptide into the lumen of the endoplasmic reticulum. The gene for protein Z4 could not be detected in the barley variety Maskin and some of its descendants. The 'high-lysine' allees, lys1 (Hiproly barley) and lys3a (Bomi mutant 1508) on chromosome 7, enhance and repress, respectively, the expression of the protein Z4 gene. Also, 1554 bp of another 8-kbp fragment of the barley genome Paz psi, similar to the protein-Z4-coding region, have been determined. Small insertions and deletions and the presence of an internal stop codon identify this fragment as part of a pseudogene related to the protein Z4 gene.     

Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1

Excitatory amino acid transporters (EAATs) are responsible for homeostasis of extracellular L-glutamate, and the glial transporters are functionally dominant. EAAT expression or function is altered in acute and chronic neurological conditions, but little is known about the regulation of EAATs in reactive astroglia found in such neuropathologies. These studies examined the effects of the bacterial endotoxin lipopolysaccharide (LPS) on glial EAATs in vitro. The effects of LPS (1 microg/ml, 24-72 h) on EAAT activity and expression were examined in primary cultures of mouse astrocytes. [(3)H]D-aspartate uptake increased to 129% of control by 72 h treatment with LPS. Saturation analysis revealed that apparent K(m) was unchanged whilst V(max) was significantly increased to 172% of control by 72 h LPS treatment. Biotinylation and Western blotting indicated that cell-surface expression of GLT-1 was significantly elevated (146% control) by LPS treatment whereas GLAST expression was unchanged. Confocal analyses revealed that LPS treatment resulted in cytoskeletal changes and stellation of astrocytes, with rearrangement of F-actin (as shown by phalloidin labelling). Immunocytochemistry revealed clustering of GLAST, and increased expression and redistribution of GLT-1 to the cell-surface following treatment with LPS. Similar experiments were conducted in microglia, where LPS (50 ng/ml) was found to up-regulate expression of GLT-1 at 24 and 72 h in concert with cytoskeletal changes accompanying activation. These findings suggest an association of cytoskeletal changes in glia with EAAT activity, with the predominant adaptation involving up-regulation and redistribution of GLT-1.     

Subunit composition of photosystem I and identification of center X as a [4Fe-4S] iron-sulfur cluster

A photosystem I (PS-I) preparation from barley (Hordeum vulgare L.) containing the reaction center protein P700-chlorophyll a-protein 1 (CP1) and smaller polypeptides with apparent molecular masses of 18, 16, 14, 9.5, 9, 4, and 1.5 kDa has been analyzed with respect to subunit stoichiometry. CP1 contains two homologous subunits with approximate masses of 82 kDa. CP1 and the smaller polypeptides were isolated, and the amino acid composition of each component and of the PS-I preparation was determined. Based on the amino acid composition data and the determined ability of each isolated polypeptide to bind Coomassie Brilliant Blue, the PS-I complex is shown to contain 1 mol of each of the homologous 82-kDa polypeptides as well as 1 mol of the 18-, 16-, 9.5-, and 9-kDa polypeptides for each mol of P700. The total polypeptide mass of the PS-I complex is 209 kDa excluding tryptophan and approximately 220 kDa including tryptophan. The two 82-kDa subunits present/P700 provide cysteine residues for binding only one Fe-S center. In conjunction with the earlier reported binding of four iron and four acid-labile sulfides to CP1/P700 (H?j, P. B., Svendsen, I., Scheller, H. V., and M?ller, B. L. (1987) J. Biol. Chem. 262, 12676-12684), this demonstrates the center X is a [4Fe-4S] cluster and eliminates the possibility of center X being composed of two [2Fe-2S] clusters.     

Chromosome 7 and 19 Trisomy in Cultured Human Neural Progenitor Cells

Background

Stem cell expansion and differentiation is the foundation of emerging cell therapy technologies. The potential applications of human neural progenitor cells (hNPCs) are wide ranging, but a normal cytogenetic profile is important to avoid the risk of tumor formation in clinical trials. FDA approved clinical trials are being planned and conducted for hNPC transplantation into the brain or spinal cord for various neurodegenerative disorders. Although human embryonic stem cells (hESCs) are known to show recurrent chromosomal abnormalities involving 12 and 17, no studies have revealed chromosomal abnormalities in cultured hNPCs. Therefore, we investigated frequently occurring chromosomal abnormalities in 21 independent fetal-derived hNPC lines and the possible mechanisms triggering such aberrations.

Methods and Findings

While most hNPC lines were karyotypically normal, G-band karyotyping and fluorescent in situ hybridization (FISH) analyses revealed the emergence of trisomy 7 (hNPC⁺⁷) and trisomy 19 (hNPC⁺¹⁹), in 24% and 5% of the lines, respectively. Once detected, subsequent passaging revealed emerging dominance of trisomy hNPCs. DNA microarray and immunoblotting analyses demonstrate epidermal growth factor receptor (EGFR) overexpression in hNPC⁺⁷ and hNPC⁺¹⁹ cells. We observed greater levels of telomerase (hTERT), increased proliferation (Ki67), survival (TUNEL), and neurogenesis (β_III-tubulin) in hNPC⁺⁷ and hNPC⁺¹⁹, using respective immunocytochemical markers. However, the trisomy lines underwent replicative senescence after 50–60 population doublings and never showed neoplastic changes. Although hNPC⁺⁷ and hNPC⁺¹⁹ survived better after xenotransplantation into the rat striatum, they did not form malignant tumors. Finally, EGF deprivation triggered a selection of trisomy 7 cells in a diploid hNPC line.

Conclusions

We report that hNPCs are susceptible to accumulation of chromosome 7 and 19 trisomy in long-term cell culture. These results suggest that micro-environmental cues are powerful factors in the selection of specific hNPC aneuploidies, with trisomy of chromosome 7 being the most common. Given that a number of stem cell based clinical trials are being conducted or planned in USA and a recent report in PLoS Medicine showing the dangers of grafting an inordinate number of cells, these data substantiate the need for careful cytogenetic evaluation of hNPCs (fetal or hESC-derived) before their use in clinical or basic science applications.     

Real-time ichthyoplankton drift in Northeast Arctic cod and Norwegian spring-spawning herring

Background

Individual-based biophysical larval models, initialized and parameterized by observations, enable numerical investigations of various factors regulating survival of young fish until they recruit into the adult population. Exponentially decreasing numbers in Northeast Arctic cod and Norwegian Spring Spawning herring early changes emphasizes the importance of early life history, when ichthyoplankton exhibit pelagic free drift. However, while most studies are concerned with past recruitment variability it is also important to establish real-time predictions of ichthyoplankton distributions due to the increasing human activity in fish habitats and the need for distribution predictions that could potentially improve field coverage of ichthyoplankton.

Methodology/Principal Findings

A system has been developed for operational simulation of ichthyoplankton distributions. We have coupled a two-day ocean forecasts from the Norwegian Meteorological Institute with an individual-based ichthyoplankton model for Northeast Arctic cod and Norwegian Spring Spawning herring producing daily updated maps of ichthyoplankton distributions. Recent years observed spawning distribution and intensity have been used as input to the model system. The system has been running in an operational mode since 2008. Surveys are expensive and distributions of early stages are therefore only covered once or twice a year. Comparison between model and observations are therefore limited in time. However, the observed and simulated distributions of juvenile fish tend to agree well during early fall. Area-overlap between modeled and observed juveniles September 1^st range from 61 to 73%, and 61 to 71% when weighted by concentrations.

Conclusions/Significance

The model system may be used to evaluate the design of ongoing surveys, to quantify the overlap with harmful substances in the ocean after accidental spills, as well as management planning of particular risky operations at sea. The modeled distributions are already utilized during research surveys to estimate coverage success of sampled biota and immediately after spills from ships at sea.     

Adsorption of HSA, IgG and laminin-1 on model hydroxyapatite surfaces--effects of surface characteristics

Ellipsometry and mechanically assisted sodium dodecyl sulphate elution was utilized to study the adsorption of human serum albumin (HSA), human immunoglobulin G (IgG), and laminin-1, as well as competitive adsorption from a mixture of these proteins on spin-coated and sintered hydroxyapatite (HA) surfaces, respectively. The HA surfaces were characterized with respect to wettability and roughness by means of water contact angles and atomic force microscopy, respectively. Both surface types were hydrophilic, and the average roughness (Sa) and surface enlargement (Sdr) were lower for the sintered compared to the spin-coated HA surfaces. The adsorbed amounts on the sintered HA increased as follows: HSA < laminin-1 < IgG < the protein mixture. For the competitive adsorption experiments, the adsorbed fractions increased accordingly: HSA < laminin-1 < IgG on both types of HA substratum. However, a higher relative amount of HSA and laminin-1 and a lower relative amount of IgG was found on the spin-coated surfaces compared to the sintered surfaces. The effects observed could be ascribed to differences in surface roughness and chemical composition between the two types of HA substratum, and could have an influence on selection of future implant surface coatings.