Claudine Chevalier

Restoration of the jejunal mucosa in rats refed after prolonged fasting.

To investigate the importance of body fuel depletion on gut rehabilitation after food deprivation, we compared the kinetics of jejunal mucosa alteration and restoration in rats that were refed after reaching different stages in body fuel depletion. Rats (P2) were refed while still in the so-called phase II, where body protein utilization is minimized, whereas rats (P3) were refed when they had reached the stage of increasing protein utilization (phase III). There was a significant decrease in total mass of intestine (P2, -30%; P3, -40%) and jejunal mucosa (P2, -52%; P3, -60%), as well in the size of the crypts (P2, -15%; P3, -36%) and villi (P2, -37%; P3, -55%). Structural changes of the mucosa included disappearance of some villi and a reduction in the size and number of crypts. Despite the larger morphological alterations in P3, the restoration of mucosa was as fast and complete after only 3 days of refeeding for both P2 and P3 rats. The respective roles of the mitosis pressure and of the lamina propria dynamics were studied. The rapid reversibility of the gut mucosal alterations due to fasting might constitute an integrative process.

Gill epithelial cells kinetics in a freshwater teleost, Oncorhynchus mykiss during adaptation to ion-poor water and hormonal treatments.

The aim of this work was to determine the kinetics of the dramatic development of the gill chloride cells (CCs) during adaptation of the salmonid Oncorhynchus mykiss to an ion-poor environment.To monitor cell division, the incorporation in the mitotic cell DNA of bromo-deoxyuridine (BrdUrd) was visualized with a monoclonal antibody. The density of labelled nuclei was used as an index of cellular division (proliferation), concomitantly with morphometry of phenotypic changes monitored with SEM.In the filament epithelium, a phase of CC differentiation occurred within 12h after the transfer, followed by a delayed phase of cell proliferation (48h). In the lamellar epithelium, the present study demonstrates the absence of cell proliferation after ion-poor water transfer. The conclusion is that proliferation (mitosis) is important in the primary filament whereas differentiation and migration (from the filament) is the main mechanism for the appearance of CCs on the secondary lamellae.The present study suggests that cortisol promoted differentiation, but not division, of cells. CCs, presumably premature, were stained by anti-cortisol monoclonal antibody indicating the presence of cortisol. No mature CCs were stained.Growth hormone (oGH, ratGH) increased the rate of cell division both in lamellar and filament epithelium.

Observations of the intestinal mucosa using environmental scanning electron microscopy (ESEM); comparison with conventional scanning electron microscopy (CSEM)

In order to evaluate the potential use of environmental scanning electron microscopy (ESEM) in biology, structural changes of the jejunal villi of rats were studied after periods of fasting and refeeding, using a conventional scanning electron microscope (CSEM) and ESEM. While observation using the CSEM, involves chemical fixation, drying and coating, observation of fresh, unprepared materials can be directly realized with the ESEM. Environmental microscopy provides a relatively new technology for imaging hydrated materials without specimen preparation and conductive coating. Direct observation of biological samples in their native state is therefore possible with an ESEM. After fasting, the jejunal mucosa is dramatically reduced in size, splits and holes appearing at the tip of the villi. These changes were observed whatever the type of technique used. Artifacts due to the sample preparation for CSEM observation (drying, coating) can therefore be excluded. However, CSEM and ESEM must be used jointly. While, CSEM must be preferred for surface analysis involving high magnifications, ESEM observation, on the other hand, can prove valuable for determining the living aspect of the samples.

Rh Proteins and NH4+ –activated Na+-ATPase in the Magadi Tilapia (Alcolapia grahami), a 100% Ureotelic Teleost Fish.

SUMMARY The small cichlid fish Alcolapia grahami lives in Lake Magadi, Kenya, one of the most extreme aquatic environments on Earth (pH ~10, carbonate alkalinity ~300 mequiv l−1). The Magadi tilapia is the only 100% ureotelic teleost; it normally excretes no ammonia. This is interpreted as an evolutionary adaptation to overcome the near impossibility of sustaining an NH3 diffusion gradient across the gills against the high external pH. In standard ammoniotelic teleosts, branchial ammonia excretion is facilitated by Rh glycoproteins, and cortisol plays a role in upregulating these carriers, together with other components of a transport metabolon, so as to actively excrete ammonia during high environmental ammonia (HEA) exposure. In Magadi tilapia, we show that at least three Rh proteins (Rhag, Rhbg and Rhcg2) are expressed at the mRNA level in various tissues, and are recognized in the gills by specific antibodies. During HEA exposure, plasma ammonia levels and urea excretion rates increase markedly, and mRNA expression for the branchial urea transporter mtUT is elevated. Plasma cortisol increases and branchial mRNAs for Rhbg, Rhcg2 and Na+,K+-ATPase are all upregulated. Enzymatic activity of the latter is activated preferentially by NH4+ (versus K+), suggesting it can function as an NH4+-transporter. Model calculations suggest that active ammonia excretion against the gradient may become possible through a combination of Rh protein and NH4+-activated Na+-ATPase function.

Intralipid 10%: Physicochemical Characterization

OBJECTIVES Parenteral fat emulsions contain two populations of particles: artificial chylomicrons rich in triacylglycerols (TAG), and liposomes (bilayer of phospholipids [PL] enveloping an aqueous phase). Centrifugation permits isolating the liposomes in the infranatant called mesophase. The aim of the present work was to better characterize this mesophase chemically and to view the particles it contains by electron microscopy. METHODS Electron microscopy (Philips 410) was performed after cryofracture on native 10% Intralipid, mesophase (centrifugation for 1 h at 27 000 g), and a liposome-enriched fraction (ring of density 1.010-1.030 g/l obtained after centrifuging mesophase in a KBr density gradient at 100 000 g for 24 h). The TAG and protein content of the mesophase was analyzed and the proteins partially characterized by immunodetection (Western-blot). RESULTS This electron microscope study of 10% Intralipid gives evidence for the coexistence of artificial chylomicrons (mean diameter, 260 nm) and liposomes (43 nm), the latter being smaller than expected and containing 8% w/w TAG after purification. The solubilization of TAG in PL bilayers (reported to be < or = 3.1% w/w) might have been increased in parenteral emulsions by the manufacturing process or/and the high TAG/PL ratio. Minute amounts of proteins have also been detected and partially characterized using a specific antibody raised against the human 7 kDa Anionic Polypeptide Factor (APF), known to strongly interact with PL in bile. CONCLUSIONS This work has shown that the size (mean diameter, 43 nm) of the liposomes present in 10% Intralipid is smaller than that usually assumed. Traces of hydrophobic proteins in the emulsion may account for certain allergic reactions sometimes observed in infused patients.

Morphological evaluation of spermatogenesis in Lake Magadi tilapia (Alcolapia grahami): A fish living on the edge

Spermatogenesis in Lake Magadi tilapia (Alcolapia grahami), a cichlid fish endemic to the highly alkaline and saline Lake Magadi in Kenya, was evaluated using light and transmission electron microscopy. Spermatogenesis, typified by its three major phases (spermatocytogenesis, meiosis and spermiogenesis), was demonstrated by the presence of maturational spermatogenic cells namely spermatogonia, spermatocytes, spermatids and spermatozoa. Primary spermatogonia, the largest of all the germ cells, underwent a series of mitotic divisions producing primary spermatocytes, which then entered two consecutive meiotic divisions to produce secondary spermatocytes and spermatids. Spermatids, in turn, passed through three structurally distinct developmental stages typical of type-I spermiogenesis to yield typical primitive anacrosomal spermatozoa of the externally fertilizing type (aquasperm). The spermatozoon of this fish exhibited a spheroidal head with the nucleus containing highly electron-dense chromatin globules, a midpiece containing ten ovoid mitochondria arranged in two rows and a flagellum formed by the typical 9 + 2 microtubule axoneme. In addition, the midpiece, with no cytoplasmic sheath, appeared to end blindly distally in a lobe-like pattern around the flagellum; a feature that was unique and considered adaptive for the spermatozoon of this species to the harsh external environment. These observations show that the testis of A. grahami often undergoes active spermatogenesis despite the harsh environmental conditions to which it is exposed on a daily basis within the lake. Further, the spermiogenic features and spermatozoal ultrastructure appear to be characteristic of Cichlidae and, therefore, may be of phylogenetic significance.

Metabolism and antioxidant defense in the larval chironomid Tanytarsus minutipalpus: adjustments to diel variations in the extreme conditions of Lake Magadi.

ABSTRACT Insect larvae are reported to be a major component of the simple but highly productive trophic web found in Lake Magadi (Kenya, Africa), which is considered to be one of the most extreme aquatic environments on Earth. Previous studies show that fish must display biochemical and physiological adjustments to thrive under the extreme conditions of the lake. However, information for invertebrates is lacking. In the present study, the occurrence of the larval chironomid Tanytarsus minutipalpus is reported in Lake Magadi for the first time. Additionally, changes in larval metabolism and antioxidant defense correlated with diel variations in the extremely hostile environmental conditions of the lake are described. Wide variations in water temperature (20.2-29.3°C) and dissolved oxygen content (3.2-18.6 mg O2 l−1) were observed at different times of day, without significant change in water pH (10.0±0.03). Temperature and dissolved oxygen were higher at 13:00 h (29.3±0.4°C and 18.6±1.0 mg O2 l−1) and 19:00 h (29.3±0.8°C and 16.2±1.6 mg O2 l−1) and lower at 01:00 h (21.1±0.1°C and 10.7±0.03 mg O2 l−1) and 07:00 h (20.2±0.4°C and 3.2±0.7 mg O2 l−1). Significant and parallel increases in parameters related to metabolism (cholinesterase, glucose, cholesterol, urea, creatinine and hemoglobin) and the antioxidant system (SOD, GPx, GR, GSH and GSSG) were observed in larvae collected at 13:00 h. In contrast, no significant changes were observed in pro-oxidants (ROS and NO), TOSC and oxidative damage parameters (LPO and DNA damage). Therefore, the observed increases in temperature and dissolved O2 content in Lake Magadi were associated with changes in the antioxidant system of T. minutipalpus larvae. Adjustments performed by the chironomid larvae were efficient in maintaining body homeostasis, as well as protecting biomolecules against oxidative damage, so that oxidative stress did not occur. GSH-GSSG and GPx-GR systems appeared to play an essential role in the adjustments displayed by the chironomid larvae during the diel changes in the extreme conditions of Lake Magadi. Summary: Insect larvae display adjustments in metabolism and oxidative status to overcome the diel variations in the extreme and harsh physicochemical conditions of Lake Magadi, a saline and alkaline lake in Kenya.

Gill epithelium ultrastructure of crayfish (Astacus leptodactylus) and immunolocalization of Na+/K+‐ATPase

Mycoplasma penetrans is a recently identified mollicute (wall-less bacteria) with unique morphology, isolated from HIV-infected patients. This mycoplasma has been found to be statistically associated with HIV infection, to be cytopathic in vitro with the capacity of penetrating into the cytoplasm of eucaryotic cells (Neyrolles O., Brenner C., Prevost M.-C., Fontaine T., Montagnier L., Blanchard A. (19981. Microbiology 144,1247-55).