The effects of osmotic tissue dehydration and air drying on morphology and energy transfer in two species of porphyra

Studies were conducted to document the effects on morphology and energy transfer in photosynthesis of severe tissue dehydration induced either by air-drying or by immersing the tissues of two Porphyra species in hyperosmotic solutions. These studies showed that the dehydration-tolerant intertidal alga, Porphyra perforata J.Ag., was almost unaffected by either of these treatments, while the dehydration-sensitive Porphyra nereocystis Anders. was damaged similary by both treatments. Damage to that sensitive species was characterized by ruptured organelles as seen by interference microscopy as well as by increased fluorescence emission at 682 nanometers emanating from allophycocyanin. These results suggest that a disruption of energy transfer between allophycocyanin and chlorophyll a occurs because of the damage to membranes following tissue dehydration, and that the increase in the yield of phycobilin fluorescence is a good indicator of these phenomena. Thus, air-drying and osmotic-dehydration appear to have similar physiological consequences in a dehydration-sensitive alga but almost no effect in a tolerant species.     

Ultrastructural organization of heterochromatin within sea urchin sperm nuclei

Chromatin within swollen or lysed isolated sperm nuclei of the sea urchin, Strongylocentrotus purpuratus, was examined by electron microscopy. Spread preparations of lysed sperm nuclei demonstrated dense aggregates of nondispersed material and beaded filaments radiating from these aggregates. These beaded fibers are similar in size and appearance to the “beads-on-a-string” seen as characteristic of chromatin spreads from numerous interphase nuclei. The beads are nucleosomes that have an average diameter of 130 Å. The interconnecting string is 40 Å indiameter and corresponds to the spacer DNA. In thin sections of swollen nuclei the sperm chromatin appears to be composed of 400 Å superbeads that are closely apposed to form 400 Å fibers. As the chromatin disperses, the superbeads are seen to be attached to one another by chromatin fibers of 110 Å diameter. In thin sections, the 400 Å superbeads appear to disperse directly into the 110 Å fibers with no intervening structures. This work demonstrates that the heterochromatin in Strongylocentrotus purpuratus sperm nuclei is composed of nucleosomes that form 100 Å filaments that are compacted into 400 Å superbeads. The superbeads coalesce to give the morphological appearance of 400 Å fibers.     

Elongation of duplex DNA by recA protein

recA protein, which is essential for the recombination process in Escherichia coli, was incubated in the presence of 5′-γ-thiotriphosphate with circular plasmid pBR_βG containing small single-stranded gaps. Stable complexes were formed which appear in the electron microscope as fibres with a diameter about five times that of naked DNA. Complex formation appears to be a co-operative process whereby the average rise per base-pair with respect to the fibre axis increases from 3·39 ± 0·08 Å to 5·20 ± 0·18 Å. The elongation of DNA by about 50% is compatible with an unwinding of the double helix and an intercalating mode of binding of recA and/or 5′-γ-thiotriphosphate to DNA.     

Biochemical and ultrastructural characterizations of nucleoprotamine in human sperm heads treated with micrococcal nuclease and salt

Preparation of human sperm heads containing mainly nucleoprotamines as their chromatin constituent was described. Acrosome-depleted human sperm heads were treated with micrococcal nuclease followed with 2 M NaCl. The treatment specifically removed nucleohistone as small chromatin fragments from the human sperm heads. The nuclease-NaCl-treated sperm head pellets primarily contained nuclease-resistant nucleoprotamine as their chromatin. Transmission electron microscopic study showed that there were two populations of the nuclease-NaCl-treated human sperm head pellets. One population had thick nucleoprotamine cords of ?650–1,200 Å in diameter, and the other had thin nucleoprotamine cords of ?330–420 Å.     

Membrane phase behavior during cooling of stallion sperm and its correlation with freezability

Abstract

Stallion sperm exhibits great male-to-male variability in survival after cryopreservation. In this study, we have investigated if differences in sperm freezability can be attributed to membrane phase and permeability properties. Fourier transform infrared spectroscopy (FTIR) was used to determine supra and subzero membrane phase transitions and characteristic subzero membrane hydraulic permeability parameters. Sperm was obtained from stallions that show differences in sperm viability after cryopreservation. Stallion sperm undergoes a broad and gradual phase transition at suprazero temperatures, from 30–10°C, whereas freezing-induced dehydration of the cells causes a more severe phase transition to a highly ordered gel phase. Sperm from individual stallions showed significant differences in post-thaw progressive motility, percentages of sperm with abnormal cell morphology, and chromatin stability. The biophysical membrane properties evaluated in this study, however, did not show clear differences amongst stallions with differences in sperm freezability. Cyclodextrin treatment to remove cholesterol from the cellular membranes increased the cooperativity of the suprazero phase transition, but had little effects on the subzero membrane phase behavior. In contrast, freezing of sperm in the presence of protective agents decreased the rate of membrane dehydration and increased the total extent of dehydration. Cryoprotective agents such as glycerol decrease the amount of energy needed to transport water across cellular membranes during freezing.     

Pf1 bacteriophage replication-assembly complex: X-ray fibre diffraction and scanning transmission electron microscopy

X-ray fibre diffraction and scanning transmission electron microscopy have been used to investigate the structure of an intracellular complex between circular single-stranded viral DNA and a viral DNA-binding protein. This complex is an intermediate between replication and assembly of the filamentous bacteriophage Pf1. By scanning transmission electron microscopy, the complex has a length of 1.00 μm and M_r = 29.6 × 10⁶. It consists of 1770 protein subunits, each of 15,400 M_r, and one viral DNA molecule of 2.3 × 10⁶M_r: there are 4.2 ± 0.5 nucleotides per subunit. The structure is flexible in solution, but in oriented dry fibres it forms a regular helix of 45 Å pitch having 6.0 dimeric protein subunits per turn, with an axial spacing of 7.5 Å between dimers and 1.9 Å between adjacent nucleotides. Model calculations suggest that the protein dimers may be oriented in a direction approximately perpendicular to the 45 Å helix, so that each dimer spans the two anti-parallel DNA chains. The results imply that conformational changes are required of the DNA as it is transferred from the double-stranded form to the replication-assembly complex, and subsequently to the virion.     

Air-Drying of Cells,the Novel Conditions for Stimulated Synthesis of Triacylglycerol in a Green Alga,Chlorella kessleri

Triacylglycerol is used for the production of commodities including food oils and biodiesel fuel. Microalgae can accumulate triacylglycerol under adverse environmental conditions such as nitrogen-starvation. This study explored the possibility of air-drying of green algal cells as a novel and simple protocol for enhancement of their triacylglycerol content. Chlorella kessleri cells were fixed on the surface of a glass fibre filter and then subjected to air-drying with light illumination. The dry cell weight, on a filter, increased by 2.7-fold in 96 h, the corresponding chlorophyll content ranging from 1.0 to 1.3-fold the initial one. Concomitantly, the triacylglycerol content remarkably increased to 70.3 mole% of fatty acids and 15.9% (w/w), relative to total fatty acids and dry cell weight, respectively, like in cells starved of nitrogen. Reduction of the stress of air-drying by placing the glass filter on a filter paper soaked in H₂O lowered the fatty acid content of triacylglycerol to 26.4 mole% as to total fatty acids. Moreover, replacement of the H₂O with culture medium further decreased the fatty acid content of triacylglycerol to 12.2 mole%. It thus seemed that severe dehydration is required for full induction of triacylglycerol synthesis, and that nutritional depletion as well as dehydration are crucial environmental factors. Meanwhile, air-drying of Chlamydomonas reinhardtii cells increased the triacylglycerol content to only 37.9 mole% of fatty acids and 4.8% (w/w), relative to total fatty acids and dry cell weight, respectively, and a marked decrease in the chlorophyll content, on a filter, of 33%. Air-drying thus has an impact on triacylglycerol synthesis in C. reinhardtii also, however, the effect is considerably limited, owing probably to instability of the photosynthetic machinery. This air-drying protocol could be useful for the development of a system for industrial production of triacylglycerol with appropriate selection of the algal species.     

Osmotic dehydration of Kappaphycus alvarezii

The red alga Kappaphycus alvarezii has been reported to be a potential raw material for functional food due to its high content of soluble dietary fibre, mineral, omega-3 fatty acids as well as a substantial amount of essential amino acids. In order to benefit from these excellent nutritional properties, this project aimed to develop a high-value dehydrated snack from K. alvarezii using osmotic dehydration (OD) treatment prior to hot air-drying. A 3?×?3 factorial design with 50°, 60° and 70°Brix sucrose concentration as well as treatment temperatures of 30, 35 and 40 °C were used. In general, an increase in sucrose concentration and temperature promoted mass transfer. OD treatment using 70°Brix sucrose concentration at 40 °C caused case hardening of the seaweed that reduced the solid gain (p?<?0.05). Firmness of the seaweed increased with sucrose concentration and was not altered by temperature (p?<?0.05). The colour of the seaweed was not affected by OD treatment (p?>?0.05), but dehydrated seaweed became darker at high sugar concentration. Interaction effect between sucrose concentration and temperature was found to affect the water loss and solid gain of the OD treatment (p?<?0.05). The best sensory acceptable dehydrated seaweed was successfully identified. The final product contained high dietary fibre and very low Na/K ratio.     

The crystal and molecular structure of tri-o-ethylamylose (tea 3)

The crystal and molecular structure of a tri-O-ethylamylose polymorph, TEA 3, has been solved by stereochemical conformation and packing analysis, combined with X-ray fibre diffraction analysis. The unit cell is orthorhombic, space group P2₁2₁2₁, with a  15.36 (±0.03) Å, b  12.18 (±0.05) Å, and c (fibre repeat)  15.48 (±0.01) Å. The actual chain conformation is a 4₃ helix with the EtO-6 group in the tg position, as was found in the polymorph TEA 1.     

Changeability of sperm chromatin structure during liquid storage of ovine semen in milk-egg yolk- and soybean lecithin-based extenders and their relationships to field-fertility

The aim of this experiment was to study the effect of semen extender on sperm chromatin structure and to correlate chromatin integrity with field-fertility of preserved ram semen. Ejaculates of at least 2 × 10⁹ sperm/ml and 70 % progressive motility were collected using an artificial vagina from Chios rams (n = 11, 4–6 years old), split-diluted to 1 × 10⁹ sperm/ml with milk-egg yolk- and soybean lecithin (Ovixcell^®)-based extenders, packaged in 0.5-ml straws and examined after 6, 24 and 48 h of storage at 5 ± 1 °C. Evaluation endpoints were computer-assisted sperm motion analysis, fluorescence-based analysis of chromatin structure by chromomycin A₃ and acridine orange assays, and 65-day pregnancy rate (PR) of 34- to 36-h preserved semen after intra-cervical insemination of ewes (n = 154) in progestagen-synchronized estrus. Neither extender nor storage time had any influence on incidence of decondensed chromatin. Unlike Ovixcell^® extender, deterioration of sperm motility (P < 0.01) and chromatin stability (P < 0.005) was detected after 48 h of storage in milk-egg yolk extender. Sperm motility accounted for 14.4–18.5 % of variations in chromatin integrity (P < 0.001). No significant difference was found in PR of Ovixcell^®- and milk-egg yolk-stored semen. Nevertheless, PR differed between rams (14.3–71.4 %; P < 0.025). Chromatin integrity explained 10.2–56.3 % of variations in PR (P < 0.05–0.01). A pronounced decline in PR (19.1 %) was observed when percentages of decondensed and destabilized chromatin have reached thresholds of 10.5–30 % and 4–9 %, respectively. In conclusion, Ovixcell^® is superior to milk-egg yolk extender in preserving chromatin stability and motility. Chromatin defects are negatively associated with sperm fertility.     

The integrity of sperm chromatin in young tropical composite bulls

Sperm chromatin fragmentation is associated with subfertility, but its relationship with age progression in young bulls is poorly understood. The objective was to assess sperm chromatin fragmentation during the early post-pubertal development of 20 tropical composite bulls, using a sperm chromatin structure assay (SCSA) and sperm-bos-halomax (SBH). Bulls were subjected to bull breeding soundness evaluation (BBSE) at mean ages of 13, 18, and 24 mo. Traits measured included liveweight (WT), body condition score (BCS) and scrotal circumference (SC). Semen samples were collected by electroejaculation and assessed for mass activity (MA), motility (Mot), concentration (conc), sperm morphology and chromatin fragmentation. Concentration (r = 0.34, P = 0.0076), Mot (r = 0.36, P = 0.0041) and percentage of morphologic normal sperm (percent normal sperm (PNS); r = 0.31, P = 0.0132) were positively correlated with age. The percentage of sperm with proximal droplets (PD) was negatively correlated with age (r = −0.28, P = 0.0348), whereas neither SCSA nor SBH results were significantly correlated with age. The percentage of sperm with chromatin fragmentation using SCSA was correlated with PNS (r = −0.53, P < 0.0001), the percentage of sperm with head abnormalities (r = 0.68, P < 0.0001) and the percentage of intact sperm (Int) with SBH (r = −0.26, P = 0.0456). In summary, for assessment of sperm chromatin fragmentation, samples could be equally collected at 13, 18 or 24 mo of age, as results did not vary with age.     

Structure of polar pili from Pseudomonas aeruginosa strains K and O

The polar pili of Pseudomonas aeruginosa strains K and O are hollow cylinders with 52 Å outer diameter and 12 Å inner diameter. There is a girdle of low electron density (interpreted as due to a local concentration of hydrophobic amino acid side-chains) centred at 31 Å diameter. Similar X-ray diffraction patterns are obtained from oriented fibres of the two types of pili, to a resolution of 7 Å in the equatorial direction and 4 Å in the meridional direction. The two types of pilin protein subunits have a similar molecular weight, and their sequences contain a number of homologous regions. They form a helical array with 4.06 to 4.08 units per turn of a basic helix that has a pitch of 40.8 Å for strain K pili and 41.3 Å for strain O pili at 75% relative humidity. A method is described for distinguishing between very similar diffraction patterns.There is strong intensity at 10 Å near the equator and at 5 Å near the meridian on the diffraction patterns. This intensity distribution is characteristic of α-helical rods running roughly in the direction of the fibre axis. The orientation of these rods was established by the fit between the transform of an α-helical polyalanine model and the strong near-equatorial layer-line.     

Polymorphism in B-DNA: X-ray diffraction studies on Li-DNA fibres

From X-ray diffraction studies it is generally believed that B-DNA has the structural parameters n = 10 and h = 3.4 Å. However, for the first time we report that polymorphism in the B-form can be observed in DNA fibres. This was achieved by the precise control of salt and humidity in fibres and by the application of the precession method of X-ray diffraction to DNA fibres. The significant result obtained is that n = 10 is not observed for crystalline fibre patterns. In fact, n = 10 and h = 3.4 Å are not found to occur simultaneously. Instead, a range of values, n = 9.6–10.0 and h = 3.35 Å–3.41 Å is observed.     

Chromatin structure analysis of spermatozoa from reciprocal chromosome translocation (RCT) carriers with known meiotic segregation patterns

The presence of reciprocal chromosome translocations (RCTs), as well as sperm chromatin disturbances, is known to exert negative influence on male fertility. The aim of this study was to identify an association between chromosome structural rearrangements in male RCT carriers and sperm seminological parameters (concentration, motility, morphology), chromatin status (fragmentation and maturity), meiotic segregation pattern and observed chromosomal hyperhaploidy. Sperm samples originated from ten male RCT carriers with reproductive failure/success. TUNEL assay (DNA fragmentation) and chromomycin A3 (CMA3)/aniline blue (AB) staining (chromatin maturity) were used to analyze sperm chromatin status while fluorescent in situ hybridization (FISH) was applied to observe meiotic segregation patterns and hyperhaploidy in spermatozoa. We found that the mean level of sperm DNA fragmentation in the RCT carrier group (18.0 ± 11.9%) was significantly higher (p = 0.0006) than the mean of the control group (7.5 ± 4.3%). There was no correlation observed between sperm DNA fragmentation levels (5.6–38.0%) and the frequency of genetically normal/balanced gametes (34.3–62.4%), sperm seminological quality or revealed reproductive failure. In contrast, a correlation between the frequencies of genetically normal/balanced spermatozoa and of gametes with mature chromatin was observed (CMA3: R = 0.4524, p = 0.2604; AB: R = 0.5238, p = 0.1827). A statistically significant increase in the hyperhaploidy level of selected chromosomes in all analyzed RCT carriers was documented but was not correlated to sperm seminology or fertility status. Further evaluation and additional assays toward sperm chromatin quality assessment in RCT carriers is suggested to explain the complexity of genomic structural rearrangements and its possible relevance to reproductive success or failure.     

Size and number of nerve fibres in the central nervous system connectives of the cockroach Blaberus craniifer

The size and number of axons in the ventral cord connectives of the cockroach Blaberus craniifer were determined from montages constructed of electron micrographs of the left connective of each of the connective pairs examined. The fibres were grouped into three main diameter categories: fine fibres from 0·2 to 1 μm, small fibres from 1 to 6 μm, and large fibres from 6 to 24 μm. In the five different left connectives examined, the fine fibres numbered from 2006 to 8535 and composed from 56·5 to 83 per cent of the total fibres. The small fibres numbered from 1269 to 2361 and composed from 16·5 to 41 per cent of the total fibre number. The large axons ranged between 29 and 220 in number which represented from 0·5 to 2·5 per cent of the fibre population.     

Constitutive Expression of Yes-Associated Protein (Yap) in Adult Skeletal Muscle Fibres Induces Muscle Atrophy and Myopathy

The aim of this study was to investigate the function of the Hippo pathway member Yes-associated protein (Yap, gene name Yap1) in skeletal muscle fibres in vivo. Specifically we bred an inducible, skeletal muscle fibre-specific knock-in mouse model (MCK-tTA-hYAP1 S127A) to test whether the over expression of constitutively active Yap (hYAP1 S127A) is sufficient to drive muscle hypertrophy or stimulate changes in fibre type composition. Unexpectedly, after 5–7 weeks of constitutive hYAP1 S127A over expression, mice suddenly and rapidly lost 20–25% body weight and suffered from gait impairments and kyphosis. Skeletal muscles atrophied by 34–40% and the muscle fibre cross sectional area decreased by ≈40% when compared to control mice. Histological analysis revealed evidence of skeletal muscle degeneration and regeneration, necrotic fibres and a NADH-TR staining resembling centronuclear myopathy. In agreement with the histology, mRNA expression of markers of regenerative myogenesis (embryonic myosin heavy chain, Myf5, myogenin, Pax7) and muscle protein degradation (atrogin-1, MuRF1) were significantly elevated in muscles from transgenic mice versus control. No significant changes in fibre type composition were detected using ATPase staining. The phenotype was largely reversible, as a cessation of hYAP1 S127A expression rescued body and muscle weight, restored muscle morphology and prevented further pathological progression. To conclude, high Yap activity in muscle fibres does not induce fibre hypertrophy nor fibre type changes but instead results in a reversible atrophy and deterioration.     

Outgrowth of nerve fibres from Drosophila central nervous system cultured in vitro

Explants of the central nervous system of Drosophila have been shown to produce nerve fibres in vitro. The effects of various culture conditions on fibre outgrowth have been examined. Nervous tissue could form nerve fibres in vitro when the explants were obtained from mid-embryonic or early- and mid-pupal stages, but not when they were obtained from larvae or late-pupae. The effect of the temperature-sensitive mutation shibire^ts has been investigated by placing mutant explants into culture at permissive (17°C) or restrictive (28°C) temperatures. No differences in the extent of fibre outgrowth between wild-type and shibire^ts were observed, regardless of the temperature of cultivation.     

Isolation, characterization and mapping of genes differentially expressed during fibre development between Gossypium hirsutum and G. barbadense by cDNA-SRAP

Gossypium hirsutum and G. barbadense are two cultivated tetraploid cotton species with differences in fibre quality. The fibre of G. barbadense is longer, stronger and finer than that of G. hirsutum. To isolate genes expressed differently between the two species during fibre development, cDNA-SRAP (sequence-related amplified polymorphism) was applied. This technique was used to analyse genes at different stages of fibre development in G. hirsutum cv. Emian22 and G. barbadense acc. 3-79, the parents of our interspecific mapping population. A total of 4096 SRAP primer combinations were used to screen polymorphism between the DNA of the parents, and 275 highly polymorphic primers were picked out to analyse DNA and RNA from leaves and fibres at different developmental stages of the parents. A total of 168 DNA fragments were isolated from gels and sequenced: 54, 30, 38 and 41 from fibres of 5, 10, 15 and 20 days post-anthesis, respectively, and five from multi stages. To genetically map these sequences, 104 sequence-specific primers were developed and were used to screened polymorphism between the mapping parents. Finally, six markers were mapped on six chromosomes of our backbone interspecific genetic map. This work can give us a primary knowledge of differences in mechanism of fibre development between G. hirsutum and G. barbadense.