Characterization of the primary sonic muscles in Carapus acus (Carapidae): a multidisciplinary approach.

Sound production in carapid fishes results from the action of extrinsic muscles that insert into the swim bladder. Biochemical, histochemical and morphological techniques were used to examine the sonic muscles and compare them with epaxial muscles in Carapus acus. Sonic fibres are thicker than red and thinner than white epaxial fibres, and sonic fibres and myofibrils exhibit an unusual helicoidal organization: the myofibrils of the centre are in a straight line whereas they are more and more twisted towards the periphery. Sonic muscles have both features of red (numerous mitochondria, high glycogen content) and white (alkali-stable ATPase) fibres. They differ also in the isoforms of the light chain (LC3) and heavy chain (HC), in having T tubules at both the Z-line and the A-I junction and in a unique parvalbumin isoform (PAI) that may aid relaxation. All these features lead to the expression of two assumptions about sound generation: the sonic muscle should be able to perform fast and powerful contractions that provoke the forward movement of the forepart of the swim bladder and the stretching and "flapping" of the swim bladder fenestra; the helicoidal organization allows progressive drawing of the swim bladder fenestra which emits a sound when rapidly released in a spring-like manner.

Effect of ophthalmic viscosurgical devices on lens epithelial cells: a morphological study.

PURPOSE: To investigate the morphological effects of Viscoat (sodium hyaluronate 3.0%-chondroitin sulfate 4.0%) on lens epithelial cells (LECs). SETTING: Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, Texas, USA, and the Laboratory of Ultrastructural Morphology, Zoological Institute, University of Liège, Liège, Belgium. METHODS: Human LECs collected via capsulorhexis were examined by light microscopy (LM) and transmission electron microscopy (TEM). Lens epithelial cells from rabbit capsulorhexis samples were studied by LM and TEM following exposure to Provisc (sodium hyaluronate 1.0%) or Viscoat ophthalmic viscoelastic device (OVD). Since Viscoat is hypertonic (340 mOsm), hypertonic, isotonic, and hypotonic solutions were compared to investigate a possible mechanism for the observed effects. The effects of Provisc and Viscoat on rabbit LECs in the intact lens were also compared. RESULTS: Human LECs gathered via capsulorhexis following exposure to Viscoat were generally thinner than control samples and often had condensed nuclei and increased intracellular vacuolization. Rabbit capsular tissue exposed in situ to Viscoat demonstrated changes similar to those seen in humans. Cells exposed to Provisc were similar to cells in untreated controls in humans and rabbits. Corneal endothelial cells exposed to either agent were unaffected. Experiments with hypertonic and hypotonic buffers induced some of the changes noted with Viscoat, but the effects were less severe. Lens epithelial cells in intact rabbit lenses exposed to Viscoat appeared similar to LECs in the control samples. CONCLUSIONS: Light microscopy and TEM of human lens capsule tissue suggest that Viscoat induces significant morphological changes in LECs during cataract surgery. The changes may underlie the improved visualization of these cells that has been reported during cataract surgery. Corneal endothelial cells were unaffected by exposure to Viscoat. Studies in a rabbit model suggest that the hyperosmolarity of Viscoat may play a partial role in the LEC changes.

Preparation of poly(D,L-lactide) nanoparticles assisted by amphiphilic poly(methyl methacrylate-co-methacrylic acid) copolymers.

When co-precipitated with amphiphilic copolymers from DMSO, poly(D,L-lactide) (PLA) can be readily converted into stable sub-200 nm nanoparticles by addition of an aqueous phase, free of any polymeric stabilizers such as poly(vinyl alcohol) or Poloxamer. In this work, the ability of random poly(methyl methacrylate-co-methacrylic acid) copolymers (PMMA-co-MA) to stabilize PLA nanoparticles was demonstrated, and the properties of PLA/PMMA-co-MA nanoparticles were investigated. When co-precipitated with PMMA-co-MA, PLA was totally converted into nanoparticles using a polymer concentration in DMSO (Cp) below 17.6 mg ml(-1), and a PMMA-co-MA proportion above a critical value depending on the content of MA repeating units (X). For instance, the lowest PMMA-co-MA proportion required was 0.9 mg mg(-1) PLA for X = 12%, and 0.5 mg mg(-1) PLA for X = 25% (for C(PLA) = 16 mg ml(-1) DMSO). The nanoparticle diameter was essentially independent of X, the proportion of PMMA-co-MA, and the PLA molecular weight, except for oligomers for which the nanoparticle diameter was smaller. It decreased when the organic phase was diluted (126 +/- 13 nm for Cp = 17.6 mg ml(-1), and 81 +/- 5 nm for C(P) = 5.6 mg ml(-1)). The time-dependence of the stability and the degradation of PLA/PMMA-co-MA nanoparticles was discussed. One of the main advantages of this technique is the ability to control surface properties and to bring functional groups to otherwise non-functionalized PLA nanoparticles. To illustrate this, a conjugate of PMMA-co-MA25 and biotin was synthesized, and used to prepare biotinylated nanoparticles that could be detected by fluorescence and transmission electron microscopy after infiltration into ligatured rat small intestine.

Immunocytochemistry of lipids: chemical fixatives have dramatic effects on the preservation of tissue lipids.

We report here the effects of chemical fixatives on lipids studied under conditions simulating the immunogold labelling of phosphatidylserine. Using anti-phosphatidylserine antibodies, it is shown that the labelling intensity of a phosphatidylserine/phosphatidylcholine coating depends largely on the conditions of fixation. In fact, the usual aldehydic fixatives washed out most of the phostphatidylserine, thus preventing the binding of anti-phosphatidylserine antibodies. This was confirmed on biological samples such as rat liver and brain by measuring the loss of radiolabelled lipids during the fixation procedure. Furthermore, the complete procedure of tissue preparation for electron microscopical observation was investigated. The loss of (radiolabelled) lipids was studied in tissue samples during fixation and resin embedding. The results showed that the classical procedure (glutaraldehyde fixation followed by epoxy resin embedding) results in the loss of 73-91% of the tissue lipids whereas in unfixed, freeze-substituted samples, more than 76% of the tissue lipids are preserved.

Human periodontal ligament fibroblast behavior on chemically conditioned dentine: an in vitro study.

BACKGROUND: Chemical root conditioning is widely used in an attempt to improve the outcome of regenerative periodontal surgery, but its effect on connective tissue cell proliferation and biosynthetic activity has been poorly studied. The goal of the present study was to test in vitro the consequences of conditioning human dentine by citric acid or minocycline on the behavior of attached human periodontal ligament (HPDL) cells in terms of proliferation, protein synthesis and morphological appearance. METHODS: HPDL cells were seeded on powdered human dentine, either untreated or conditioned for 3 minutes with 3% citric acid or 2.5% minocycline HCI. Scanning (SEM) and transmission (TEM) electron microscopic observations were performed, and 3H-thymidine and 3H-proline incorporation tests were used to evaluate the proliferative and the biosynthetic activities. RESULTS: Cell spreading was already evident and the penetration of cytoplasmic processes into dentinal tubules were frequently observed on all dentine types after 2 hours of attachment. After 24 hours of incubation, citric acid conditioning promoted an intense spreading of the cells, while minocycline HCI conditioning induced the formation of a dense feltwork of cellular processes. HPDL fibrolasts adherent to both types of surface-conditioned dentine exhibited a significantly higher rate of proliferation (P<0.01) as well as a significantly higher level of total protein and of collagen synthesis (P<0.01) than on untreated dentine. CONCLUSIONS: These data suggest that during periodontal surgery a conditioning of the root surface by citric acid or by minocycline HCI could promote the attachment, the proliferation, and the biosynthetic activity of HDPL, prerequisites to periodontal regeneration.

Microencapsulation of erythromycin and clarithromycin using a spray-drying technique.

Previous methods of microencapsulation are unable to process particles smaller than 100 microm without organic solvents or the use of multistep processes. The present study investigates the feasiblity of a one-step spray-drying process to microencapsulate erythromycin and clarithromycin, antibiotics known to have an unpleasant, bitter taste. Mixtures of clarithromycin (5% by weight) or erythromycin (30% by weight) with a biodegradable polymer were prepared and spray-dried under specific conditions of temperature and turbine speed. This process resulted in the microencapsulation of 80% of each drug as determined by high pressure liquid chromatography. Particle size ranged from 1 to 80 microm as determined by electron microscopy. These data show that microencapsulation of macrolides using a spray-drying technique is feasible. Spray-drying microencapsulation might be useful in the formulation of palatable oral suspensions of bitter tasting drugs.

In vitro stimulation of human gingival epithelial cell attachment to dentin by surface conditioning.

BACKGROUND: Chemical root conditioning is widely used to improve the outcome of regenerative periodontal therapies by favoring the attachment of the regenerated periodontal structures. Although the effect of root conditioning on periodontal mesenchymal cells is well documented, very little is known about its potential effect on the re-formation of the junctional epithelium, a crucial event for the protection of the wound. The goal of the present study was to test in vitro the consequences of dentin conditioning with citric acid or minocycline on the attachment kinetics and morphology of human gingival keratinocytes (HGK). METHODS: The attachment kinetics of HGK to samples of powdered human dentin (particle size 44 to 76 microm) were examined by use of 3H-labeled cells. The morphology of attached epithelial cells was then determined by scanning electron microscopy (SEM). RESULTS: When the initial adhesion kinetics of cells on untreated dentin were tested, the percentage of attached HGK proved to be dependent on the number of plated cells and the time of incubation (from 0 to 12 hours). Conditioning the dentin by 3% citric acid or by minocycline-HCl (at 0.01, 0.1, or 2.5%) significantly increased (P <0.005) keratinocyte attachment beyond 6 hours, without notable differences between the 2 substances at any concentration. The attachment kinetics of HGK preincubated for 24 hours by 10 microg/ml minocyline-HCl on untreated dentin was found to be similar to that observed for non-preincubated cells. These results are in agreement with the SEM observations: indeed, the surface conditioning of dentin significantly modified the morphology of attached HGK, whereas the preincubation of these cells with minocyline-HCl did not. CONCLUSIONS: These results suggest that minocycline-HCl does not exert a direct effect on human gingival epithelial cells. In contrast, conditioning the dentin by citric acid or by minocycline stimulates the attachment of HGK, which could lead to a rapid periodontal healing by favoring the re-formation of a junctional epithelium.

Comparison between the natural postnatal maturation and the spermine-induced maturation of the rat intestine.

In the suckling rats, orally provided spermine induced structural and biochemical changes in the intestine, which are characteristics of the postnatal maturation. This induced maturation was compared to that occurring spontaneously. Eight mumol spermine were administered orally once a day, for one or three days, to suckling rats which were 11 days old at the beginning of the experiment. The animals were killed 0, 2, 4, 6, 8, 10 hours or 3 days after the first treatment. Control rats from the same litter were treated in the same way but received only the vehicle. In order to complete the study of the naturally occurring maturation, another group of rats was killed when they were 12, 13, 15, 16, 17, 18, 19, 20, 21 or 30 days old. Animal and intestine weights were measured. Disaccharidase specific activity, and protein, DNA and RNA contents were estimated in the small intestine. Histological and ultrastructural aspects of the intestinal mucosa were examined. For all these parameters, the maturation induced by spermine ingestion appeared close to that occurring naturally at weaning. Consequently, dietary spermine induces all the morphological and biochemical modifications characterizing the intestinal postnatal maturation in the suckling rat suggesting a role of the polyamines in the naturally occurring processes.

In vitro modulation of human gingival epithelial cell attachment and migration by minocycline-HCL.

Although the influence of tetracyclines on periodontal connective tissue cells has been the topic of many in vitro and in vivo studies, data regarding their effects on gingival epithelial cells are scarce. The present in vitro study was designed to examine the influence of minocycline, a semi-synthetic analog of tetracycline, on human gingival keratinocyte (HGK) attachment and migration. Attachment tests were performed with HGK prelabeled by tritiated amino-acids. Increasing concentrations of minocycline (10, 50, 100 micrograms/ml) in the medium produced no significant modification of cell adhesion kinetics compared to control conditions, except for 100 micrograms/ml which statistically significantly (p < 0.05) reduced the number of attached cells beyond 6 h. A 24-h cell preincubation in 10 micrograms/ml of minocycline did not alter the kinetics of HGK attachment. Scanning electron microscopic observations of attached HGK showed that the presence of 10 micrograms/ml of minocycline in the "attachment medium" induced the production of multiple filopodial extensions. Migration tests in Boyden chambers for 40 h demonstrated that HGK preincubation for 24 h in a 10 micrograms/ml minocycline-HCl solution increased significantly (p < 0.005) cell migration towards a gradient of fetal calf serum. The presence of 10 micrograms/ml of minocycline in contact with the keratinocytes in the upper compartment of the migration chambers also produced a significant (p < 0.005) result. In contrast, the presence of minocycline in the lower compartments did not produce any chemoattractive effect. Within the limits of their significance, these results suggest that, at concentrations not beyond 50 micrograms/ml, minocycline could fasten the periodontal wound coverage by epithelial cells and allow the normal reformation of a junctional epithelium.

Fine structural survey of old cuticle degradation during pre-ecdysis in two European Atlantic crabs.

Light and transmission electron microscopy were used to monitor changes due to the degradation of the old exoskeleton and related events in the sclerites, articular membranes, and gills of two decapod crustaceans (Carcinus maenas and Macropipus puber) during pre-ecdysis. In both sclerites and articular membranes, degradation follows a similar general pattern in both crab species, while the gill cuticle appears unaltered. In early pre-ecdysis (D(0)), the degradation of the old cuticle starts with the secretion of ecdysial droplets by the epidermis. Apolysis, occurring at stage D(1)', is re-defined as an event, not necessarily morphologically observable, consisting in the loss of adherence between the epidermis and the old cuticle during early pre-ecdysis of arthropods. At the stage D(1)''', the moulding of the epidermal cell surface occurs in preparation to the deposition of the new cuticle and causes the opening of the ecdysial cleft. In the principal layer of sclerites, degradation of the chitin-protein microfibres should precede mineral dissolution. In contrast to the other degraded cuticle layers, the membranous layer of sclerites and the innermost endocuticular lamellae of articular membranes are transformed into a digestion-resistant fibrous network resembling the ecdysial membrane of insects.

"Exolysosomes," enzyme-containing vesicles in the ecdysial space of molting crabs.

Free vesicle-like bodies (VLBs) present in the ecdysial space of cuticle regions undergoing degradation during preecdysis of the Atlantic shore crab Carcinus maenas have been interpreted either as infectious organisms or as secretion structures associated with degradation of the old cuticle. Ultrastructural, cytochemical, and immunocytological investigations were performed to test these hypotheses and to see whether VLBs are peculiar to this crab species. Similar VLBs were systematically found in two other preecdysial crabs, Cancer pagurus and Macropipus puber. In Car, maenas, they originate during early premolt inside Golgi buddings and are often gathered into large vacuoles in epidermal cells. The histochemical azo-dye technique and a cerium-based cytochemical method revealed acid phosphatase activity in both the ecdysial space and the VLBs, while Feulgen's method and immunocytological labeling always failed to reveal any DNA or RNA in either the ecdysial space or the VLBs. We conclude that VLBs are not infectious organisms but "extracellular" cuticle-degrading organelles of lysosomal origin and propose to coin them "exolysosomes."

Scintigraphic visualization of intrathecal liposome biodistribution.

BACKGROUND: Liposomes containing local anaesthetics have been administered intrathecally and in the epidural space. Poor attention has been given to the pharmacokinetics of liposomes as drug carriers. Therefore, we observed the biodistribution of liposomes after intrathecal injection in rats by scintigraphic imaging during 24 h. METHODS: We administered 99mTc-labeled multilamellar (MLV) and small unilamellar vesicles (SUV) of defined size and volume dispersities into the cerebrospinal fluid at the lumbar level. Those vesicles were free of contamination by radiolabeled colloids as visualized by light and electron microscopy and of neurotoxic products from phosphatidylcholine hydrolysis and peroxidation, both during the preparation process and after 24 h incubation in cerebrospinal fluid at 37 degrees C in vitro. RESULTS: SUV immediately diffused from the lumbar site of injection to the head and were cleared between 1 and 24 h after injection. MLV were cleared more slowly from the spinal space and appeared in the head region 1 h after injection where they accumulated up to 24 h. These differences were explained in terms of vesicle sizes and volumes. SUV with 0.05 micron diameters were rapidly absorbed into the blood through the arachnoid granulations. In contrast, particles larger than the upper size limit of the arachnoid granulations permeability (+/- 8 microns) could accumulate in the head with a slow elimination rate. CONCLUSION: This difference in clearance from the intrathecal space outlines the importance of defining the size of the liposomes, the distribution of a tracer or a drug inside the liposomal preparation, the chemical stability and the absence of toxic degradation products of liposome formulations before clinical use.

Cytochemical investigations on tunic morphogenesis in the sea peach Halocynthia papillosa (Tunicata, Ascidiacea) 2: demonstration of proteins.

In a previous paper, cellulose fibres were demonstrated in the larval, the metamorphosing, and the juvenile tunics. In this paper we used cytochemical methods and X-ray microanalysis to obtain additional information on tunic morphogenesis in Halocynthia papillosa. The chemical composition of the tunic evolves with its structural complexity. The larval and juvenile fibres are shown to be structurally and chemically different. While neither proteins nor glycosaminoglycans seem to be associated with the larval fibres, the juvenile fibres consist of a cellulose core wrapped in a sheath of tannophilic proteins. Patches of glycosaminoglycans line their longitudinal axes. In the course of metamorphosis, the cuticle undergoes profound modifications in regions of spine morphogenesis. Granular material that was previously called fibro-granular material (Lübbering et al., 1993) is essential to the formation of cuticular plates and spines. During metamorphosis, this material accumulates in epidermal granules and is discharged into the tunic. It crosses the fundamental layer of the tunic and reaches the cuticle. Our results strongly suggest that this material consists of proteins rich in cysteine and hydrophobic amino acids.

99mTechnetium-stannous oxinate as marker of liposome formulations.

Liposomes associated with tin(II) dioxinate were prepared from egg yolk phosphatidylcholine and cholesterol as sterile and pyrogen-free multilamellar or unilamellar vesicles. Complexing of liposomal tin(II) dioxinate with 99mTc attained 98% of the added radioactivity. Thirty percent 99mTc were released during 24-h incubation in biological fluids. The absence of tin colloids seen by electron microscopy and the stability of liposomal phospholipid and tin(II) dioxinate during 72-h incubation at 37 degrees C in plasma and cerebrospinal fluid would allow safe and reliable scintigraphic liposome pharmacokinetic studies.

Cytochemical investigations on tunic morphogenesis in the sea peach Halocynthia papillosa (Tunicata, Ascidiacea). 1: demonstration of polysaccharides.

The distribution of carbohydrates was demonstrated in the embryonic, larval, and juvenile tunics of Halocynthia papillosa. An enzyme-gold marker (cellobiohydrolase-Au) was used to identify cellulose on ultrathin sections. This is the first time this biopolymer has been detected in the embryonic or larval tunic of an ascidian. Cellulose is present from the initial tail-bud stage onwards, as soon as the outer compartment of the tunic appears. Both compartments of the larval tunic also contain non-cellulosic polysaccharides, as demonstrated by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method. Our observations point to two types of cellulose synthesis. One occurs during the embryonic and larval stages, when glycogen-like material is stored in epidermal intracellular lacunae and discharged into the tunic where it is presumably used to synthesize cellulose throughout the depth of the tunic. The second occurs from the onset of metamorphosis onwards, just above the apical plasmalemma of epidermal cells, like cellulose biogenesis in plants.

Ultrastructural modifications in cultured fetal quail hepatocytes exposed to pesticides and PCBs.

There is increasing interest in cultured hepatocytes as a tool for solving toxicological and pharmacological problems while reducing laboratory animal experimentation. In the present study, fetal hepatocytes from the Japanese quail (Coturnix coturnix japonica) were used as an in vitro alternative model for evaluating the effects of PCBs and various pesticide-type chemicals on cell ultrastructure. Major alterations were demonstrated. The most striking effects of toxicants were an increase in the number of cisternae of the rough endoplasmic reticulum (RER), various alterations of mitochondrial morphology, a decreased glycogen content, vacuolization of the cytoplasm, and the appearance of concentric membrane arrays (CMA's), also called myelin-like figures. Other changes were sometimes observed, such as altered cell junctions, an increased lipid content, deformations of the nuclei, or the appearance of crystalline structures. These ultrastructural modifications seem to be dose-dependent. The present in vitro findings are validated by similar observations previously made in vivo on Japanese quail. They confirm the effectiveness of this technique as a biomonitoring tool for the evaluation of environmental quality. Yet the multiplicity of possible toxic effects, even for xenobiotics of a same category, makes it necessary to screen additional indicators of toxicity, such as the detoxifying activity of monooxygenases.

Biodistribution of liposomes after extradural administration in rodents.

We have mapped over 24 h the biodistribution of 99mTc-labelled multilamellar and small unilamellar liposomes in rabbits and rats by scintigraphic imaging after extradural injection. Multilamellar vesicles formed a depot at the site of injection; small unilamellar vesicles spread immediately along the extradural space and entered the systemic compartment 30 min after injection. Well-delineated liver and kidney labellings were seen after 24 h. The use of 3H-cholesterol-labelled small unilamellar vesicles suggested hepatic capture of intact liposomes with sizes averaging 0.05 microns drained unmodified into the systemic circulation through the extradural lymphatics. These results have led to the selection of multilamellar vesicles (0.1-15 microns size range) for clinical trials using liposome-associated local anaesthetics.

Biodistribution of liposome-associated bupivacaine after extradural administration to rabbits.

After one extradural injection of 0.25% bupivacaine 0.3 ml and 3H-bupivacaine 0.005 mCi in multilamellar liposomes, no systemic radioactivity (plasma, liver, heart muscle) was obtained for 1 h, and the labelling was less than that of systemic distribution of plain bupivacaine for the following 3 h. In contrast, radioactivity in the lumbar spinal nerves peaked in the first hour and remained higher than that of plain bupivacaine for 4 h. No radioactivity was measured in cerebrospinal fluid. Small unilamellar vesicles incorporating 3H-cholesterol did not significantly label spinal nerves and central nervous structures indicating that the mode of action of liposomal bupivacaine did not involve uptake by nerve structures. Rapid uptake of radioactivity by spinal nerves suggested exchange of bupivacaine between liposomes and nerve sheaths.

Immunogold labelling of fatty acyl chains.

For the first time, antibodies against a hydrophobic hapten have been used for immunogold labelling of a lipid antigen (BSA-C18:1 conjugate) coated on polystyrene. The labelling was visualised either directly in transmission electron microscopy or in light microscopy after silver enhancement. Good recognition of the fatty acyl chain was obtained even after treatment of the antigen coat with various cross-linking fixatives used for electron microscopy, i.e. formaldehyde, glutaraldehyde and osmium tetroxide.