Ultrasound and microbubble-targeted delivery of therapeutic compounds

The molecular understanding of diseases has been accelerated in recent years, producing many new potential therapeutic targets. A noninvasive delivery system that can target specific anatomical sites would be a great boost for many therapies, particularly those based on manipulation of gene expression. The use of microbubbles controlled by ultrasound as a method for delivery of drugs or genes to specific tissues is promising. It has been shown by our group and others that ultrasound increases cell membrane permeability and enhances uptake of drugs and genes. One of the important mechanisms is that microbubbles act to focus ultrasound energy by lowering the threshold for ultrasound bioeffects. Therefore, clear understanding of the bioeffects and mechanisms underlying the membrane permeability in the presence of microbubbles and ultrasound is of paramount importance. (Neth Heart J 2009;17:82-6.)     

Effects of evodiamine and rutaecarpine on the secretion of corticosterone by Zona fasciculata‐reticularis cells in male rats

Evodiamine (EVO) and rutaecarpine (RUT) are two bioactive alkaloid isolated from Chinese herb named Wu–Chu–Yu. Previous studies have shown that EVO and RUT possess thermoregulation, vascular regulation, anti‐allergic, anti‐nociceptive and anti‐inflammatory activities. The mechanisms of EVO and RUT effect on steroidogenesis are not clear. The goal of this study was to characterize the mechanism by which EVO and RUT affect corticosterone production in rat zona fasciculata‐reticularis (ZFR) cells. ZFR cells were isolated from adrenal glands of male rats and incubated with adrenalcorticotropin (ACTH, 10^?9 M), forskolin (an adenylyl cyclase activator, 10^?5 M), 8‐bromo‐adenosine 3′:5′‐cyclic monophosphate (8‐Br‐cAMP, a permeable cAMP analog, 10^?4 M), or steroidogenic precursors including 25‐hydroxycholesterol, pregnenolone, progesterone, and deoxycorticosterone, 10^?5 M each, in the presence or absence of EVO and RUT respectively (0–10^?3 M) at 37°C for 1 h. The concentrations of corticosterone, pregnenolone and progesterone in the media were measured by radioimmunoassay. After administration of ZFR cells with EVO or RUT (10^?4 M) for 60 and 120 min, Western blot analysis was employed to explore the influence of EVO and RUT on the expression of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR). EVO and RUT reduced both basal and ACTH‐, forskolin‐, as well as 8‐Br‐cAMP‐stimulated corticosterone production in rat ZFR cells. The enhanced corticosterone production caused by the administration of four steroidogenic precursors was decreased following EVO or RUT challenge. These results suggest that EVO and RUT inhibit corticosterone production in rat ZFR cells via a mechanism involving: (1) a decreased activity of cAMP‐related pathways; (2) a decreased activity of the steroidogenic enzymes, that is, 3β‐hydroxysteroid dehydrogenase (3β‐HSD) and 11β‐hydroxylase (P450c11), during steroidogenesis; and (3) an inhibition of StAR protein expression. J. Cell. Biochem. 108: 469–475, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of catechin,epicatechin and epigallocatechin gallate on testosterone production in rat leydig cells

Catechins have been reported to have many pharmacological properties such as the effects of anti‐oxidative, anti‐inflammatory, anti‐carcinogenic, anti‐ultraviolet, and reduction of blood pressure as well as glucose and cholesterol levels. However, the effect of catechins on the reproductive mechanism is still unknown. In the present study, the effects of catechins on testosterone secretion in rat testicular Leydig cells (LCs) were explored. Both in vivo and in vitro investigations were performed. Purified LCs were incubated with or without catechin (CCN), epicatechin (EC), epigallocatechin gallate (EGCG, 10^?10–10^?8 M) under challenge with human chorionic gonadotropin (hCG, 0.01 IU/ml), forskolin, SQ22536 (an adenylyl cyclase inhibitor), 8‐bromo‐adenosine 3′:5′‐cyclic monophosphate (8‐Br‐cAMP), A23187 (a calcium ionophore), and nifedipine (10^?5 M), respectively. To study the effects of catechins on steroidogenesis, steroidogenic precursors‐stimulated testosterone release was examined. The functions of the steroidogenic enzymes including protein expression of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory (StAR) protein were investigated and expressed by Western blotting. Catechins increased plasma testosterone in vivo in male rats. In vitro, low‐dose concentration of catechins increased gonadotropin releasing hormone (GnRH)‐stimulated luteinizing hormone (LH) release by anterior pituitary gland and hCG‐stimulated testosterone release by LCs of male rats. These results suggested that catechins stimulated testosterone production by acting on rat LCs via the mechanism of increasing the action of cAMP, but not P450scc, StAR protein or the activity of intracellular calcium. EC, one of the catechins increased the testosterone secretion by rat LCs via the enzyme activities of 17β‐hydroxysteroid dehydrogenase (17β‐HSD). J. Cell. Biochem. 110: 333–342, 2010. © 2010 Wiley‐Liss, Inc.     

Visual discrimination between two sexually deceptive <Emphasis Type="Italic">Ophrys</Emphasis> species by a bee pollinator

Almost all species of the orchid genus Ophrys are pollinated by sexual deception. The orchids mimic the sex pheromone of receptive female insects, mainly hymenopterans, in order to attract males seeking to copulate. Most Ophrys species have achromatic flowers, but some exhibit a coloured perianth and a bright, conspicuous labellum pattern. We recently showed that the pink perianth of Ophrys heldreichii flowers increases detectability by its pollinator, males of the long-horned bee Eucera berlandi. Here we tested the hypothesis that the bright, complex labellum pattern mimics the female of the pollinator to increase attractiveness toward males. In a dual-choice test we offered E. berlandi males an O. heldreichii flower and a flower from O. dictynnae, which also exhibits a pinkish perianth but no conspicuous labellum pattern. Both flowers were housed in UV-transmitting acrylic glass boxes to exclude olfactory signals. Males significantly preferred O. heldreichii to O. dictynnae flowers. In a second experiment, we replaced the perianth of both flowers with identical artificial perianths made from pink card, so that only the labellum differed between the two flower stimuli. Males then chose between both stimuli at random, suggesting that the presence of a labellum pattern does not affect their choice. Spectral measurements revealed higher colour contrast with the background of the perianth of O. heldreichii compared to O. dictynnae, but no difference in green receptor-specific contrast or brightness. Our results show that male choice is guided by the chromatic contrast of the perianth during the initial flower approach but is not affected by the presence of a labellum pattern. Instead, we hypothesise that the labellum pattern is involved in aversive learning during post-copulatory behaviour and used by the orchid as a strategy to increase outcrossing.     

Intestinal crypt proliferation. II. Computer modelling of mitotic index data provides further evidence for lateral and vertical cell migration in the absence of mitotic activity

The position-dependent mitotic index before, and 1, 2 and 3 h after vincristine was scored. The accumulation of cells in mitosis leads to an increase in the mitotic index from 0.06 to 0.34 at crypt positions 8-12. Surprisingly, the leading edge of the position-related mitotic index distribution moves to higher crypt positions although cell division was stopped. In addition, the vertical clustering of mitotic figures in sections was recorded. The data were examined using a previously described computer crypt model. We conclude: the average mitotic phase duration is about 0.7 h (40 min) and varies little with cell position; the geometrical correction factor for overscoring mitoses in crypt sections is about 0.6-0.7 and adjacent cell columns can merge. Lateral cell displacement after mitosis, as predicted in a previous model analysis, would be a mechanism to counteract other forces that tend to reduce the crypt circumference. In the normal steady state merging and expansion processes would just balance each other. This would not follow if one mechanism was blocked. Thus we propose a new concept in which the crypt geometry would be dynamically determined by cell proliferative activity in connection with lateral positioning of new cells on one hand and contracting forces on the other hand.     

The spermatogenesis of Ephydatia fluviatilis (Porifera)

Summary Spermatogensis of the freshwater sponge Ephydatia fluviatilis begins with differential mitosis of choanocytes in the flagellated chambers. The spermatogonia thus produced aggregate in the mesenchyme by amoeboid movement. They are surrounded by dendritic cells which eventually form the walls of spermatocysts, within which spermatogenesis takes place. Notable features are the early formation of flagella in first order spermatocytes and the appearance of two flagella in the metaphase of the second maturation division.Spermatids and spermatozoa are arranged in characteristic ways. The spermatids have their heads near the spermatocyst wall, with the flagella pointing toward the center. Later the spermatozoan heads form a median band in the spermatocyst, and the flagella extend toward the periphery.Normally all stages of spermatogenesis can be found simultaneously in a given sponge, but development within a single spermatocyst proceeds in relative synchrony. Young spermatocysts can take up additional free spermatogonia; moreover, spermatocysts at about the same stage of development, up to a maximal diatmeter of 200 m, can fuse with one another.The E. fluviatilis studied here seem to be gonochorists.     

Scoring Mitotic Activity In Longitudinal Sections of Crypts of the Small Intestine

Abstract. Various counts have been made of the number of mitotic figures in whole crypts and sections of crypts of the small intestine of the mouse. Samples were analysed from animals killed at different times of the day and at different times after administration of vincristine. Measurements have been made of the size of mitotic and interphase nuclei and of the radial position of mitotic figures. the correction factor, f, which is required to take into account the enhancement of mitotic counts in sections as a consequence of their centripetal position has been investigated. the results indicate the following: (1) transverse sections of the crypt differ from longitudinal sections if they involve cutting the intestime before fixation which may result in a relaxation of the crypt and its widening by 25%; (2) columnar cell nuclei have a shape that resembles a sphere flattened so that the average diameter is 20% greater in crypt transverse sections; (3) mitotic nuclei tend to be about half-way between the crypt edge and the central axis of the crypt; (4) between about four and seven times more mitotic figures have their mitotic axis parallel to the long axis of the crypt; (5) about one-third of all mitotic figures in a crypt are seen in a longitudinal section of the crypt. If this is related to the number of cells in the crypt as a whole and in a section, a correction factor f_d for the mitotic index of 0.59 is obtained; (6) the correction factor f_T derived from the shape and position of the mitotic figures measured in 3 μm longitudinal sections is 0.53; (7) relating cell cycle and mitotic accumulation data using a computer-based model of the crypt also permits a correction factor f_mod to be estimated. This gives a value of 0.66. When sectioned material is used to calculate a mitotic index the most appropriate correction factor is f_D; for mouse small intestine it is 0.59.     

Evolutionswege zum Larvalauge der Insekten -Die Stemmata der höheren Dipteren und ihre Abwandlung zum Bolwig-Organ

Evolutionary pathways to the larval eyes of insects. Higher Dipteran stemmata and the evolutionary development of Bolwig's organ 1. A cornrehensive morphological study of the photoreceptors in the so-called hemi — Institut für Biologie I (Zoologie) der Albert-Ludwigs-Universität, Albertstr. 21a, D-7800 Freiburg i. Brsg., FRGand acephalic lrvae of Brachycera was undertaken. In Brachyceran larvae the head casule originally is more or less retracted into the thorax. The larval hotoreceptor in Musca and Drosophila, here called Bolwig's organ, is situated on the outer side of the cephalopharyngeal apparatus, well below the surface. The aim of this study is to elucidate the homology of Bolwig's organ, i. e. whether Bolwig's organ originated from typical stemmata during the evolution of Cyclor-rhaphan larval organlsation or whether it represents a unique type of hotoreceptor. Larval photoreceptors and the anatomy of the head capsules of representative Erachycerans were examined by means of light and electron microscopy. In particular, the site and the structure of the photofecetors of Stratiomys (Stratiomyidae, Solva (Solvidae), Atherix ibis (Athericidae), Rhagio (Rhagionidae), Thereva (derevidae), Lonchoptera (Lonchoteridae), Epistrohe balteata, Volu-cella bombylans, Eristalis tenax (Syrphidae), Drosophila (f;rosophilidae), jannia and Musca domestica (Muscidae) were investigated. 2. Brachyceran stemmata are either situated on the ocular plates of the free remnant of the head (Stratiomyidae, Therevidae), or on the inner wall of an epidermal invaination, which includes epithelia of the head as well as of the thorax (Tabaniformia). In adfition, they can be found on the outer side of the tentorial phragmata (Cyclorrhapha). In all taxa studied, stemmata keep contact directly with the epithelium or through short processes of eithelial cells. 3. Ancestral Brachycera have fused stemmata that correspond with typical larval eyes of other holometabolan insects. 4. A cornea is found solely in Stratiomyidae and Xylophagidae, in contrast to all other taxa, where no dioptric apparatus is found. 5. In “Orthorrapha” and in “Aschiza”, rhabdomeric photoreceptors occur, forming a fused, star-shaped or Ktticed rhabdom. In Schizophoran larvae, retinular cells are distinguished by a different type of surface enlargement of the photoreceptor cell membrane. Here, the membrane forms flat ramella. The latter originate from transformed and/or rudimentary rhabdomeric microvilli. 6. In the primitive “Orthorrhapha”, pigment cups can be found, that are composed of retinular pirnent, whereas in derived Brachycera, pigment grains are absent. In Cyclorrhapha, tentorial piramata and their optic deressions operate as external and functional piment cups 7. In Therevidae and in Syrphidae, a tracheal tapetum lucidum can be founfi which la, been evolved independently in both families. The tapetum is always found together with inversely orientated retinular cells. 8. The homology of Brachceran stemmata is shown by a transformation series of stem-mata's site and fine structure. The homology is corroborated by the identical innervation in primitive and derived taxa, the identical site within Cyclorrhaha, and structural similarities of “Orthorrhaphan” and primitive Cylorrhaphan stemmata. Although transformations of the fine structure appear as a sequence or reductions, particular adaptations such as multilication of retinular celfs (Epistrophe), or deepening of the pigment cup (Musca) have evolved separately. Despite the fact that in Brachyceran larvae the head has been reorganized, the eyes still keep contact with the epithelium of the frontal sacks and/or tentorial phragmata. 9. The transformation series given shows that Bolwig's organ represents a highly modified stemma.     

Ultrastructural changes in hatching-gland cells of pike embryos (Esox lucius L.) and evidence for their degeneration by apoptosis

Summary Around hatching, when the pike embryo sheds its acellular egg envelope, marked changes occur in the cellular covering of the embryo. This cellular covering consists of a peridermal layer and a mono-layered presumptive epidermis. The periderm begins to disintegrate shortly before hatching and is sloughed off in the first posthatching period. The cellular covering produces hatching enzyme, the protease that partly dissolves the zona radiata interna of the acellular envelope. By means of the peroxidase-anti-peroxidase staining method with antibodies against hatching enzyme the cells producing this enzyme (hatching gland cells, HGCs) could be identified ultrastructurally. They are interspersed as single cells between the periderm and the presumptive epidermis. The secretory cycle of the HGC was studied. Hatching enzyme is released by an exocytotic secretory process in which multiple secretion into a secretion vacuole predominates. Exocytosis into surrounding intercellular spaces also occurs. These results show that the HGCs are merocrine glands. The HGC also has some holocrine nature, however, in that only a single, massive release of its secretory product occurs. The death of the transitory HGCs in posthatching stages is characterized by condensation of the cell, formation of surface protuberances and splitting up into globular cell fragments. Eventually these fragments are ingested by epidermal cells and digested. These results lead to the conclusion that the pike HGCs degenerate by apoptosis, unlike true holocrine cells.