Uranyl Acetate Staining Research Articles

Phosphate and tension homology-induced kinase 1/Parkin signaling mediates cognitive dysfunction in sepsis-associated encephalopathy through activation of hippocampal mitochondrial autophagy

To investigate the effects of gene of phosphate and tension homology (PTEN)-induced putative kinase 1 (PINK1)/Parkin pathway on hippocampal mitophagy and cognitive function in mice with sepsis-associated encephalopathy (SAE) and its possible mechanism. A total of 80 male C57BL/6J mice were randomly divided into Sham group, cecal ligation puncture (CLP) group, PINK1 plasmid transfection pretreatment groups (p-PINK1+Sham group, p-PINK1+CLP group), empty vector plasmid transfection control group (p-vector+CLP group), with 16 mice in each group. The mice in CLP groups were treated with CLP to reproduce SAE models. The mice in the Sham groups were performed laparotomy only. Animals in the p-PINK1+Sham and p-PINK1+CLP groups were transfected with PINK1 plasmid through the lateral ventricle at 24 hours before surgery, while mice in the p-vector+CLP group were transfected with the empty plasmid. Morris water maze experiment was performed 7 days after CLP. The hippocampal tissues were collected, the pathological changes were observed under a light microscope after hematoxylin-eosin (HE) staining, and the mitochondrial autophagy was observed under a transmission electron microscopy after uranyl acetate and lead citrate staining. The expressions of PINK1, Parkin, Beclin1, interleukins (IL-6, IL-1β) and microtubule-associated protein 1 light chain 3 (LC3) were detected by Western blotting. Compared with the Sham group, CLP group mice in Morris water maze experiment had longer escape latency, shorter target quadrant residence time, and fewer times of crossing the platform at 1-4 days. Under the light microscope, the hippocampal structure of the mouse was injured, the neuronal cells were arranged in disorder, and the nuclei were pyknotic. Under the electron microscope, the mitochondria appeared swollen, round, and wrapped by bilayer or multilayer membrane structures. Compared with the Sham group, CLP group had higher expressions of PINK1, Parkin, Beclin1, LC3II/LC3I ratio, IL-6 and IL-1β in hippocampus, indicating that sepsis induced by CLP could activated inflammatory response and caused PINK1/Parkin-mediated mitophagy. Compared with the CLP group, p-PINK1+CLP group had shorter escape latencies, spent more time in the target quadrant and had more number of crossings in the target quadrant at 1-4 days. Under the light microscope, the hippocampal structures of mice was destroyed, the neurons were arranged disorderly, and the nuclei were pyknotic. Under transmission electron microscope, swollen and rounded mitochondria and mitochondrial structure wrapped by double membrane or multilayer membrane structure were observed. Compared with the CLP group, the levels of PINK1, Parkin, Beclin1 and LC3II/LC3 ratio in the p-PINK1+CLP group were significantly increased [PINK1 protein (PINK1/β-actin): 1.95±0.17 vs. 1.74±0.15, Parkin protein (Parkin/β-actin): 2.06±0.11 vs. 1.78±0.12, Beclin1 protein (Beclin1/β-actin): 2.11±0.12 vs. 1.67±0.10, LC3II/LC3I ratio: 3.63±0.12 vs. 2.27±0.10, all P < 0.05], while the levels of IL-6 and IL-1β were significantly decreased [IL-6 protein (IL-6/β-actin): 1.69±0.09 vs. 2.00±0.11, IL-1β protein (IL-1β/β-actin): 1.11±0.12 vs. 1.65±0.12, both P < 0.05], suggesting that overexpression of PINK1 protein could further activate mitophagy and reduce the inflammatory response caused by sepsis. There was no statistically significant difference in the above pathological changes and related indicators between Sham group and p-PINK1+Sham group, CLP group and p-vector+CLP group. PINK1 overexpression can further activate CLP-induced mitophagy by upregulating Parkin, thereby inhibiting inflammation response and alleviate cognitive function impairment in SAE mice.

Ultrastructural characterization of the putative defensive glands (warts) in the sessile, colonial rotifer Sinantherina socialis (Gnesiotrocha; Flosculariidae)

Female Sinantherina socialis are freshwater, sessile, colonial rotifers that possess two pairs of distinctive glands (warts) located below the corona. Previous studies demonstrated that colonies are unpalatable to many invertebrate and vertebrate predators; those authors suggested that the warts were a possible source of a chemical deterrent to predation. Here we explore wart ultrastructure and cytochemisty to determine whether the warts function as exocrine glands and if their contents display any allomone-like chemistry, respectively. Externally, the warts appear as elevated bulges without pores. Internally, the warts are specialized regions of the integumental syncytium and therefore acellular. The lipid stain Nile Red labels all four warts. Two lipid membrane probes (sphingomyelin and phosphatidylinositol) also bind the warts and may be staining internal secretion vesicle membranes. In fact, wart ultrastructure is defined by hundreds of membrane-bound secretion vesicles packed tightly together. The vesicles are mostly electron-lucent and crowded into a well-defined cytoplasmic space. The cytoplasm also contains abundant ribosomes, rough endoplasmic reticulum, mitochondria, and Golgi, but nuclei are generally positioned peripheral to the packed vesicles. Absence of muscles around the warts or any signs of direct innervation suggests expulsion of gland contents is forced by general body contraction. A single specimen with ‘empty’ warts implies that secretions are released en masse from all glands simultaneously. The identity of the chemical secretion remains to be determined, but the lack of osmium and uranyl acetate staining suggests a low abundance or absence of phenols, unsaturated lipids, or NH2 and –COOH groups. This absence, combined with the positive Nile Red staining, is interpreted as evidence that vesicles contain saturated fatty acids such as lactones that are unpalatable to predators.

Toward Visualizing Genomic DNA Using Electron Microscopy via DNA Metallization

Electron microscopy‐based DNA imaging is a powerful tool that provides a high resolution for observing genomic structures involved in biochemical processes. The first method, heavy metal shadow casting, was developed in 1948. Uranyl acetate has been widely used for DNA electron microscopic imaging since the 1960s. However, for this method, scientists must deal with government regulations for the safety and disposal. Additionally, sample preparation is often complicated and time‐consuming. Recently, nanoparticles and nanowires have emerged as a new way of imaging DNA molecules under both transmission and scanning electron microscopes. However, as this technology is still in its early stages, there is room for further development. In this review, heavy metal staining, nanoparticle staining, and nanowire growth for DNA visualization are introduced. The applications of shadow casting and uranyl acetate staining in the visualization of DNA structures and protein–DNA complexes are discussed. Then, nanomaterial‐based DNA staining methods are covered, including electrostatic interactions, DNA chain modification, reducing‐group‐modified DNA ligands and DNA–peptide/protein interactions. This review provides up‐to‐date information on different DNA staining approaches and their applications in DNA studies. Ultimately, it offers a new direction for genome analysis through DNA visualization.

Proximal sesamoid bone microdamage is localized to articular subchondral regions in Thoroughbred racehorses, with similar fracture toughness between fracture and controls.

To determine whether proximal sesamoid bone (PSB) microdamage and fracture toughness differ between Thoroughbred racehorses sustaining PSB fracture and controls. Cadaveric case-control. Twenty-four Thoroughbred racehorses (n=12 PSB fracture, n=12 control). Proximal sesamoid bones were dissected, and gross pathological changes and morphological measurements were documented. High-speed exercise history data were evaluated. Microdamage was assessed in fracture, fracture-contralateral limb (FXCL) and control PSBs using whole bone lead uranyl acetate (LUA) staining with micro-CT imaging or basic fuchsin histological analysis. Fracture toughness mechanical testing was carried out in 3-point-bending of microbeams created from PSB flexor cortices. Data were analyzed using ordinal logistic and linear regression models. Microdamage was detected most commonly in the articular subchondral region of PSBs via LUA micro-CT and basic fuchsin histology. There were no differences in microdamage between FXCL and control PSBs. Fracture toughness values were similar for FXCL (1.31 MPa√m) and control (1.35 MPa√m) PSBs. Exercise histories were similar except that horses sustaining fracture spent a greater percentage of their careers in rest weeks. Microdamage was detected in the articular region of PSBs but was not greater in horses sustaining catastrophic PSB fracture. Fracture toughness of PSB flexor cortices did not differ between FXCL and control PSBs. Although uncommon, microdamage is localized to the articular region of Thoroughbred racehorse PSBs. Catastrophic PSB failure is not associated with lower PSB flexor cortex fracture toughness.

Novel electron microscopic staining method using traditional dye, hematoxylin

Uranyl acetate (UA) has been routinely used as a staining solution for ultrathin sections used in biological electron microscopy. As a radioactive nuclear material, UA is subject to strict international regulations. To develop an alternative and easy-to-use staining method for ultrathin sections, we examined various commercial light microscopic dyes. We found that Mayer’s hematoxylin followed by Reynold’s lead citrate solution showed staining results comparable to UA and Reynold’s lead citrate solution, and this method is therefore suggested as a reliable and promising alternative to UA staining.

How to stain nucleic acids and proteins in Miller spreads.

The spreading technique proposed by Miller and Beatty in 1969 allowed for the first time the visualization at transmission electron microscopy of nucleic acids and chromatin in an isolated and distended conformation. This approach is beneficial since it can reveal many aspects of chromatin organization and function that otherwise can only be indirectly inferred by biochemical methods. The final step of staining chromatin spreads is critical because it can strongly influence the interpretation of the results. We evaluated different staining techniques, and almost all provided a good result. Specifically, well-contrasted micrographs were obtained when staining with H3PW12O40 (phosphotungstic acid, PTA), as originally proposed by Miller and Beatty, and with two alternatives proposed here: uranyl acetate or terbium citrate. Quite a good contrast of the spread DNA could also be achieved using osmium ammine; while no or little contrast of nucleic acids was observed by staining with KMnO₄ (potassium permanganate) and H3PMo12O40 (phosphomolybdic acid, PMA) respectively.

SUN-032 Investigation of endotoxin cumulation in kidney tissue of rats with experimental sepsis model and the protective effect of ascorbic acid in the same model

The aim of this study was to evaluate the renal histopathology and to investigate the protective effect of ascorbic acid in rats with sepsis model. 24 young male Albino Wistar rats were divided into 4 groups (n = 6): Control (C), Ascorbic acid (AA), Lipopolysaccharide (LPS), Ascorbic acid-treated LPS group (AALPS). All groups were fed ad libitum. The AA and AALPS group were given 150 mg/kg ascorbic acid (Abdi İbrahim® Pharmaceutical Ind., Istanbul, Turkey) by gavage for 3 days. On the third day, 4 mg/kg intraperitoneal LPS (Escherichia Coli [E.coli] O 111: B4 from Sigma Aldrich) was administered to the LPS and AALPS groups 2 hours after the last dose of ascorbic acid; C and ASA groups received intraperitoneal 4 ml of physiological saline. After 6 hours, the animals' abdomens were opened under abdominal anaesthesia (Xylazine HCl 5 mg/kg and Ketamine HCl 35mg / kg) and blood samples were taken from their hearts for biochemical analysis. The kidneys were then removed and placed in 4% formaldehyde solution for light microscope (LM) and 3% glutaraldehyde solution for electron microscope (EM). Biochemistry assays: CRP, NGAL, TNFα, Presepsin (SinoGeneClon Biotech, Hangzhou, China) were examined in serum and tissue, Creatinine, Phosphorus, Ca, Cl, K, Na, Uric acid, Urea in serum. Histopathological evaluation was performed by Hematoxylin-eosin staining on an LM, uranyl acetate and pure lead citrate staining on an EM and Anti-E. coli LPS antibody (1∶100, Abcam, Cambridge, UK) on immunohistochemistry (IHC). Statistical analysis was performed by One Way Anova test and Kruskal-Wallis method using SPSS v22.0. Biochemical results are summarized in Table 1 and 2. CRP, Presepsin, NGAL, TNFα, creatinine, urea, phosphorus, calcium, chloride, potassium values showed a significant difference between the groups. LM: Histopathological examination revealed normal renal tissue in LM in the C and AA groups (Fig. 1 A). In the LPS group in LM increased blood flow in the cortical layer, loss of normal histological appearance and excessive dilation of the proximal and distal tubules are observed (Fig. 1 B; C). LPS IHC staining showed a no staining in C and AA group (Fig 2 A; C), but in LPS group increase of LPS expression in the tubular basement membrane (Fig 2 B). However, the AALPS group were showed minimal LPS expression and LM changes than LPS group (Fig. 2 D and Fig 1 D ). EM: Normal EM is observed in the C and AA group (Fig. 3A; B; Fig 4 A; B). Significant changes were made in capillary loops, endothelial basement membranes, and podocytes in ultrastructural images from the LPS group. All three layers of the basement membranes have lost their boundaries (Fig. 5 A, B and C white arrow). Mitochondrial cristae smoothing and growth in the volume of lysosomes are repeated in many endothelial cells (Fig.5 D red arrow). The most striking feature is the presence of lymphocytes (Fig.5 E white arrow). AA LPS group have weak swelling, weak hydrophilic degeneration was observed in the tissues compared with the LPS group (Figure 6A is marked with a red arrow 6B normal IV type collagen). In our study, a sepsis model was formed in accordance with the literature and the protective effect of ascorbic acid was observed in the AALPS group. Endotoxemia causes serious damage to kidney tissue. These changes may be due to renal hypoperfusion and the direct toxic effect of endotoxin.

Olfactory ensheathing cells promote nerve regeneration and functional recovery after facial nerve defects.

Olfactory ensheathing cells from the olfactory bulb and olfactory mucosa have been found to increase axonal sprouting and pathfinding and promote the recovery of vibrissae motor performance in facial nerve transection injured rats. However, it is not yet clear whether olfactory ensheathing cells promote the reparation of facial nerve defects in rats. In this study, a collagen sponge and silicone tube neural conduit was implanted into the 6-mm defect of the buccal branch of the facial nerve in adult rats. Olfactory ensheathing cells isolated from the olfactory bulb of newborn Sprague-Dawley rats were injected into the neural conduits connecting the ends of the broken nerves, the morphology and function of the regenerated nerves were compared between the rats implanted with olfactory ensheathing cells with the rats injected with saline. Facial paralysis was assessed. Nerve electrography was used to measure facial nerve-induced action potentials. Visual inspection, anatomical microscopy and hematoxylin-eosin staining were used to assess the histomorphology around the transplanted neural conduit and the morphology of the regenerated nerve. Using fluorogold retrograde tracing, toluidine blue staining and lead uranyl acetate staining, we also measured the number of neurons in the anterior exterior lateral facial nerve motor nucleus, the number of myelinated nerve fibers, and nerve fiber diameter and myelin sheath thickness, respectively. After surgery, olfactory ensheathing cells decreased facial paralysis and the latency of the facial nerve-induced action potentials. There were no differences in the general morphology of the regenerating nerves between the rats implanted with olfactory ensheathing cells and the rats injected with saline. Between-group results showed that olfactory ensheathing cell treatment increased the number of regenerated neurons, improved nerve fiber morphology, and increased the number of myelinated nerve fibers, nerve fiber diameter, and myelin sheath thickness. In conclusion, implantation of olfactory ensheathing cells can promote regeneration and functional recovery after facial nerve damage in rats.

Correlated light and electron microscopy observations of the uterine epithelial cell actin cytoskeleton using fluorescently labeled resin-embedded sections

In order to perform correlative light and electron microscopy (CLEM) more precisely, we have modified existing specimen preparation protocols allowing fluorescence retention within embedded and sectioned tissue, facilitating direct observation across length scales. We detail a protocol which provides a precise correlation accuracy using accessible techniques in biological specimen preparation. By combining a pre-embedding uranyl acetate staining step with the progressive lowering of temperature (PLT) technique, a methacrylate embedded tissue specimen is ultrathin sectioned and mounted onto a TEM finder grid for immediate viewing in the confocal and electron microscope. In this study, the protocol is applied to rat uterine epithelial cells in vivo during early pregnancy. Correlative overlay data was used to track changes in filamentous actin that occurs in these cells from fertilization (Day 1) to implantation on Day 6 as part of the plasma membrane transformation, a process essential in the development of uterine receptivity in the rat. CLEM confirmed that the actin cytoskeleton is disrupted as apical microvilli are progressively lost toward implantation, and revealed the thick and continuous terminal web is replaced by a thinner and irregular actin band, with individually distinguishable filaments connecting actin meshworks which correspond with remaining plasma membrane protrusions.

English

Bacteriophages (or phages) are parasites that infect specific bacteria. This host-selectivity can be useful to identify bacterial contaminants in food, water, environment etc. In the present study, phage was isolated from stagnant water and cultivated by overlay method against host bacteria that is E. coli. Phage titer was calculated to be 107 pfu/ml using 10-fold dilutions. Plaque reaction activity was observed within 4 to 6 h against host bacteria by spot test. Morphological identification of phage by transmission electron microscopy (TEM) using uranyl acetate staining revealed about 78 nanometers (nm) in wide phage capsid and tail length (527 nm). The isolated phage was classified into order Caudovirales since it possessed a long non-contractile tail and icosahedral capsid head, thus is a member of the family Siphoviridae. Also, the phage identified followed a lytic life cycle since plaque reaction activity was observed within 4-6 h against host bacteria. Gold immuno-functionalization using self-assembled monolayers (SAM) has been widely used for the detection of small targets, but there are limited reports available describing the detection systems for bacteria by using phages. Thus, in order to develop a suitable detection system for identification of specific bacteria, it is suitable to exploit the close association and selectivity between bacteria and bacteriophage. Bacteriophages were immobilized onto gold surface by SAM using a stable acyl amino ester intermediate generated by 1-ethyl-3(3-dimethylaminoproply) carbodiimide (EDC) and N-hydrosuccinimide (NHS) to condense the bacteriophage. Fourier transformation infrared (FTIR) microscopy presence of different functional groups present in each layer formation. Bacteriophage immobilization over the gold surface was verified through by scanning electron microscope (SEM). Electrochemical analysis was performed for a rapid and specific detection of E. coli cell. The present bio-sensing system comprises of quick and specific detection of host bacteria and possesses a very low detection limit (104 cfu/ml). We propose phages utilization as a bio-component in biosensor development for bacteria capture. Key words: Bacteriophage, biosensors, electrochemical, microscopy, immune-functionalisation and Self-assembled molecular monolayer.

Assessment of alkali–silica reaction damage through quantification of concrete nonlinearity

A newly developed nondestructive evaluation technique, Nonlinear Impact Resonance Acoustic Spectroscopy (NIRAS), is applied to concrete specimens in an ongoing assessment of aggregate alkali reactivity during standard concrete prism testing. NIRAS measures the nonlinearity in a specimen caused by the inception and growth of microcracks throughout the sample and debonding at the aggregate/cement interface. NIRAS is used to exploit the nonlinear effect of excitation amplitude dependent resonance frequency changes, which are related to nonlinearity measurements of concrete samples cast with aggregates of varying reactivity. To relate microstructural changes to changes in nonlinearity and expansion, sample characterization is performed with uranyl-acetate staining. The results demonstrate the utility of NIRAS for not only assessing the potential for ASR under standardized test conditions, but for more general damage characterization in concrete and assessment of “job mixtures.” NIRAS can distinguish reactive from nonreactive aggregates without ambiguity, as supported by sample characterization results.

Saccharomyces cerevisiae Dmc1 and Rad51 Proteins Preferentially Function with Tid1 and Rad54 Proteins, Respectively, to Promote DNA Strand Invasion during Genetic Recombination

The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.

Preparation and Characterization of pH Sensitive Microgels with Semi-Interpenetrating Polymer Network Structure

The microgels with semi-interpenetrating polymer network (semi-IPN) structure based on cross-linked poly(N-isopropylacrylamide) (PNIPAM) and linear poly(acrylic acid) (PAA) were fabricated by one-step precipitation polymerization technique. The chemical composition of the microgels was determined by Fourier transformation infrared spectroscopy and elemental analysis. It was found that the PAA content inside the microgels decreased as the pH value of reaction media increased. The semi-IPN structure of the microgels was confirmed by transmission electron microscopy using uranyl acetate staining method. The pH dependent hydrodynamic diameters obtained by dynamic light scattering revealed that these microgels can reversibly shrink and swell in the pH range from 4.0 to 6.0. The semi-IPN microgels synthesized at pH 3.0 could undergo about 120 times volume change in the pH range.

Two Distinct Amyloid β-Protein (Aβ) Assembly Pathways Leading to Oligomers and Fibrils Identified by Combined Fluorescence Correlation Spectroscopy, Morphology, and Toxicity Analyses

Nonfibrillar assemblies of amyloid β-protein (Aβ) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using Aβ labeled at the N terminus or Lys(16), we uncovered two distinct assembly pathways. One leads to highly toxic 10-15-nm spherical Aβ assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ∼330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ∼128 kDa (∼32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys(16)-labeled peptide disturbed fibril formation because the Aβ(16-20) region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying Aβ assembly steps at which inhibition may be beneficial.

PH/temperature dual stimuli-responsive microcapsules with interpenetrating polymer network structure

The microcapsules with interpenetrating polymer network (IPN) structure based on crosslinked poly (N-isopropylacrylamide) (PNIPAM) and crosslinked poly (acrylic acid) (PAA) were fabricated in a three-step process. Firstly, silica/PNIPAM core/shell composite particles were synthesized by thermo-initiated seed precipitation polymerization using 3-(trimethoxysilyl)propyl methacrylate modified silica colloidal particles as seeds and N-isopropylacrylamide and N,N′-methylenebisacrylamide (MBA) as monomer and crosslinker, respectively. Secondly, PAA network was incorporated into the shell of the composite particles by redox-initiated polymerization of acrylic acid and MBA entrapped in the PNIPAM network. Finally, the silica core of the composite particles was removed using hydrofluoric acid under certain condition to produce the microcapsules. The chemical compositions, their mass ratio, and particle sizes of the particles formed in each step were determined by Fourier transformation infrared spectroscopy, thermogravimetry, and dynamic laser light scattering (DLLS), respectively. The IPN structure of the microcapsules was identified by transmission electron microscopy (TEM) using uranyl acetate staining method, and their hollow structure was evidenced by TEM and scanning electron microscopy. Their temperature- or pH-dependent hydrodynamic diameters were measured by DLLS, and the results showed that the microcapules had both pH- and temperature-responsive properties, and the temperature-responsive component and the pH-responsive component inside the microcapsule shell had little interference with each other.

Suberized Cell Walls of Cork from Cork Oak Differ from Other Species

Plants have suberized cells that act as protective interfaces with the environment or between different plant tissues. A lamellar structure of alternating dark and light bands has been found upon transmission electron microscopy (TEM) observation of cork cells and considered a typical feature of the suberized secondary wall. We observed cork cells from periderms of Quercus suber, Quercus cerris, Solanum tuberosum, and Calotropis procera by TEM after uranyl acetate and lead citrate staining. A lamellated structure was observed in S. tuberosum and C. procera but not in Q. suber and Q. cerris where the suberized cell wall showed a predominantly hyaline aspect with only a dark dotted staining. Removal of suberin from Q. suber cells left a thinner secondary wall that lost the translucent aspect. We hypothesize that the species' specific chemical composition of suberin will result in different three-dimensional macromolecular development and in a different spatial location of lignin and other aromatics. A lamellated ultrastructure is therefore not a general feature of suberized cells.

Effects of Qichu Fujin Recipe on regeneration and repair of injured sciatic nerve in rats

To explore the effects of Qichu Fujin Recipe (QCFJR), a compound traditional Chinese medicine, in repairing sciatic nerve injury in rats. A total of 60 male Sprague-Dawley (SD) rats were randomly divided into four groups: normal control group, untreated group, mecobalamin group and QCFJR group. Except the normal control group, sciatic nerve injury was induced by crushing of left sciatic nerve. Rats in the mecobalamin group were intragastrically administered with mecobalamin solutions 150 microg/(kg x d), and rats in the QCFJR group were intragastrically administered with QCFJR 35.2 g/(kg x d), while rats in the untreated group were intragastrically administered with normal saline (0.5 mL/d), once a day for 4 weeks respectively. Sciatic function index (SFI) was determined by walking tract analysis 1 week and 2 and 4 weeks after crushing. Five rats in each group were sacrificed for histological observation at the different time points. The remnant rate of gastrocnemius wet weight and the diameter of gastrocnemius cells were calculated. Expression of S-100 protein in the distal stump of injured nerve was observed by using immunohistochemical method. Distal injured sciatic nerves were determined with toluidine blue staining and observed under a light microscope 1 week and 2 and 4 weeks after operation. Diameter of axon and depth of myelin sheath were calculated by an image analysis system. Ultrastructure of nerve fibers was determined with uranyl acetate and lead citrate staining and observed under an electron microscope. Compared with the normal control group, SFI, remnant rate of gastrocnemius wet weight, diameter of gastrocnemius cells, expression of S-100, diameter of axon and depth of myelin sheath in the mecobalamin group and the QCFJR group were significantly decreased at different time points (P<0.05), superior to those of the untreated group (P<0.05, P<0.01). Except the remnant rate of gastrocnemius wet weight and the diameter of gastrocnemius cells, there were no significant differences in other indexes between the mecobalamin group and the QCFJR group 1 week and 2 and 4 weeks after crushing (P>0.05). QCFJR can stimulate the regeneration and repair of nerve fiber and delay the skeletal muscle atrophy after sciatic nerve injury in rats.

Structure of Loricae and Stalks of Several Bacterivorous Chrysomonads (Chrysophyceae): Taxonomical Importance and Possible Ecological Significance

Stalks and loricae of chrysomonads were studied by fluorescence microscopy employing Calcoflor White-Evans blue (CW-Eb) staining, and by uranyl acetate staining of dried, whole mount preparations for electron microscopy. These structures were composed of microfibrils approximately 4 nm in diameter embedded in a matrix. The organization of the loricae of Poterioochromonas malhamenesis, "Amimonas minuta", Poterioochromonas stipitata and Ochromonas gloeopara showed a similar structural plan, consisting of a foot, stalk and cup region that together resemble a wine glass. CW-Eb-stained, microfibrillar stalks were identified also in Paraphysomonas vestita, Anthophysa vegetans and "Felimonas flocculans". These results suggest that CW-Eb-stained structures composed of microfibrils approximately 4 nm in diameter may be more common in chrysomonads than previously recognized. In cultures, these structures participate in the formation of cell aggregates and attachment of cells to substrates, and thus may be of ecological importance. Additionally, non-siliceous, CW-Eb-stained cysts were identified for the first time in P. malhamensis.

Fibrinogen Storage Disease Caused by Aguadilla Mutation Presenting With Hypobeta‐lipoproteinemia and Considerable Liver Disease

Fibrinogen Storage Disease Caused by Aguadilla Mutation Presenting With Hypobeta‐lipoproteinemia and Considerable Liver Disease

Electron microscopy of chromatin elimination in Cidaria (Lepidoptera).

Elimination chromatin observable in the middle of the bivalents in oogenesis of the geometrid moth species Cidaria variata (subgenus Thera) differs distinctly from the chromosomal chromatin by being less electron dense after uranyl acetate staining. Elimination chromatin is tightly packed in between the bivalent halves already at metaphase I, and is bursting out from between the bivalents leaving the centre of the bivalents as a hollow cavity. There is a distinct tendency for elimination chromatin to stick adjacent bivalents together. In the electron microscope it seems to have an unorganized, fibrillar appearance. The thickness of the fuzzy fibers is in the range of 100 a. Three possible hypotheses have been raised to account for the elimination of ribonucleoprotein material from chromosomes in lepidopteran oogenesis.