Histogenesis of the unique morphology of proboscidean ivory

The chequered pattern (often called Schreger pattern), which can be seen by unaided eye on transverse profiles of several proboscidean tusks and which can be emphasized by the spreading pattern of the cracks or by mineral discoloration, is an autapomorph feature of the clade Elephantoidea. The pattern differs among proboscidean taxa; therefore, it allows the coarse differentiation of elephants, mammoths, and some other basal groups. Such identification methods could facilitate efforts concerned with protection of the remaining elephant populations through ivory trade restrictions, since the tooth dentine from extinct Mammuthusprimigenius and from extant Loxodontaafricana and Elephasmaximus are the most common raw materials of the ivory carvings. The aim of this study was to show the internal structure of proboscidean ivory and to revise the existing theories on the aforementioned pattern of the elephantoids with reflections on the events which lead to the development of this microstructure. Thin sections and natural crack surfaces with various orientations of M.primigenius, Elephasantiquus, Prodeinotherium, and Deinotherium tusk fragments were used to produce a three‐dimensional model which explains the features on all profiles. The “phase shift” model is introduced, which assumes a sinusoid undulation of the dentinal tubules in radial profiles in the case of elephantoids. The model was confirmed by photomicrographs, scanning electron microscopic images, interpretation of natural crack surfaces, and radial displacement analysis of the dentinal tubules. The latter proved that the adjacent waves are not in the same phase. Several new nondestructive distinguishing methods are described here on the basis of the correlation between some microscopic and macroscopic features related to the Schreger pattern. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Stage-specific localization of cytoskeletal actin mRNA in murine seminiferous tubules and intestinal epithelia as demonstrated by in-situ hybridization

Summary In-situ hybridization experiments have been performed using isoactin ( and )-specific riboprobes in various tissues of the rat and mouse. Distribution of the grains of actin mRNAs for both and types was similar throughout sections of the rat testis. Although both mRNAs were evenly distributed in the seminiferous tubule, extremely heavy labeling was observed in about 10% of the seminiferous tubules that could be identified as stage XII of spermatogenesis. At high magnification, grains of the mRNA were found in the cytoplasm of elongating spermatids and in the Sertoli cell cytoplasm at the adluminal side. Much higher density of the grains of mRNA was observed in the neck region of the spermatids at stage XII. Thus, the dense distribution of cytoskeletal actin mRNAs is stage-specific in the tubule during spermatogenesis in the rat. The high expression of both and actin mRNAs was also observed in the epithelial cells of the intestinal crypts.     

Cellular architecture of the lamina propria of human seminiferous tubules

Summary The lamina propria of human seminiferous tubules is composed of 5 to 7 cellular layers separated by laminae of extracellular connective-tissue components. By means of immunocytochemical methods the different nature of the cellular layers could be defined for the first time. Based on the light-microscopic demonstration of both desmin-like and vimentin-like immunoreactivity in the inner 3 to 4 layers of the lamina propria, these cells can be identified as myofibroblasts. The outermost one or two cellular layers, on the contrary, only show a vimentin-like immunoreactivity indicating the pure fibroblastic nature of these cells. Therefore, the outermost cellular layers are suggested to be derivatives of the interstitium. In cases of disturbed spermatogenesis, the lamina propria is frequently considerably thickened by an increase in the extracellular matrix components between the cellular layers. Whereas the ultrastructural localization of laminin-, collagen type-IV- and fibronectin-like immunoreactivity remains unaffected in the thickened lamina propria, the desmin-like immunoreactive cells of the inner layers strongly decrease in number and staining intensity. Most probably, the myofibro-blasts lose their myoid characteristics to participate in the secretion of increased amounts of extracellular matrix components, which in turn presumably block the mediation of the lamina propria between the interstitium and the germinal epithelium. It is still unclear whether the thickened lamina propria provokes the disturbance of spermatogenesis or vice versa.     

Metabolic adaptation of the renal carbohydrate metabolism. I. Effects of starvation on the gluconeogenic and glycolytic fluxes in the proximal and distal renal tubules

Summary The influence of starvation on renal carbohydrate metabolism was studied in the proximal and distal fragments of the nephron. Starvation induced a double and opposite adaptation mechanism in both fractions of the renal tubule. In renal proximal tubules, the gluconeogenic flux was stimulated progressively during a period of 48 hours of starvation (2.15 fold), due, in part, to a significant increase in the fructose 1,6-bisphosphatase and phosphoenolpyruvate carboxykinase activities although with different characteristics. Fructose 1,6-bisphosphatase activity from this tubular fragment increased only at subsaturating subtrate concentration (68%) which involved a significant decrease in the Km (35%) for fructose 1,6-bisphosphate while there was no change in Vmax. This behaviour clearly indicates that it is related to modifications in the activity of the preexistent enzyme in the cell. Proximal phosphoenolpyruvate carboxykinase activity increased proportionally at both substrate concentrations (86 and 89% respectively) which brought about changes in Vmax without changes in Kin, all of which are in accordance with variations in the cellular levels of the enzyme. In the renal distal tubules, the glycolytic capacity drastically decreased throughout the starvation time. At 48 hours 65% of inhibition was shown. We have found a short term regulation of phosphofructokinase activity by starvation which involves an increase in Km (2.2 fold) without changes in Vmax, as a result of these kinetic changes, an inactivation of phosphofructokinase was detected at subsaturating concentration of fructose 6-phosphate. On the contrary, this nutritional state did not modify the kinetic behaviour of renal pyruvate kinase. Finally, neither proximal glycolytic nor distal gluconeogenic capacities and related enzymes activities were changed during starvation.     

The endosome-lysosome system in the absorptive cells of goldfish hindgut

Summary The vacuolar system in the absorptive cells of the goldfish hindgut was studied by rapid freeze-substituted and cytochemical techniques. The apical cytoplasm of the absorptive cells contained two types of vacuoles: endosomes and lysosomes. The former were characterized by an absence of acid phosphatase activity, a dot-like distribution of material at the peripheral rim, the labelling of the inner surface with horseradish peroxidase (HRP), and by frequent connections to cytoplasmic tubules (CT), which were also free of acid phosphatase activity. The latter vacuole was preferentially located in the deeper cytoplasm and was characterized by the presence of acid phosphatase activity, an electron-dense interior matrix, a peripheral electron-lucent region (a halo), and by the detachment of HRP from the inner surface. Connections between CTs and these latter vacuoles were rarely seen. In the deeper cytoplasm, fusion between endosomes and lysosomes was sometimes observed. These results suggest that the vacuoles which are associated with CTs are endosomes, but not lysosomes, and that internalized materials are transported through the endosome-lysosome system to a giant food vacuole in the cell.     

Computer-aided three-dimensional reconstructions of the arrangement of primary spermatocytes in human seminiferous tubules

Summary With the use of a digital image-processing method three-dimensional reconstructions of the arrangement of spermatocytes in human seminiferous tubules were performed. With this method it was possible to investigate the cellular distribution in the tubule in nearly any given perspective and projection. In addition, by means of simple mathematical procedures, such as by transformation of Cartesian coordinates into cylindrical coordinates, it was possible to vary the shape of a reconstruction, i.e., to convert the cylindrical image of a tubular portion into a right-angled r--z-representation.The present work not only confirms the existence of a complex helical plan of organization of the human seminiferous epithelium but also provides further aspects of the phenomenon of physiological germ-cell loss and its integration into the kinetics of spermatogenesis.Dedicated to Prof. E.C. Roosen-Runge, Seattle, on the occasion of his 75th birthday     

Effect of Na+, K+, ouabain,amiloride and ethacrynic acid on the transepithelial potential across Malpighian tubules of Locusta

A transepithelial potential of +8.74±0.29 mV (n = 85) has been recorded across the Malpighian tubules of Locusta. The effect of varying the Na⁺ and K⁺ concentration in the bathing medium on the transepithelial potential has been determined. The data show that the transepithelial potential does not obey the Nernst equation for K⁺. Ouabain, ethacrynic acid and amiloride all inhibit the transepithelial potential. The results are discussed in relation to the nature of the mechanisms of cation transport across the Malpighian tubules.     

Metabolic adaptation of renal carbohydrate metabolism. V.In vivo response of rat renal-tubule gluconeogenesis to different diuretics

We have studied the effects of the diuretics mersalyl, furosemide and ethacrynic acid on renal gluconeogenesis in isolated rat-kidney tubules and on the activities of the most important gluconeogenic and glycolytic enzymes in both fed and fasted rats. Mersalyl (15 mg.kg^–1 animal weight) significantly decreased the rate of gluconeogenesis in well-fed rats (68%) as well as in 24 and 48-h fasted ones (33 and 37% respectively). This inhibition occurred when lactate, pyruvate, glycerol or fructose were used as substrates. Ethacrynic acid at a dose of 50 mg.kg^–1 animal weight provoked a transient inhibition of renal glucose production by almost 20% but only in fed rats with lactate as substrate, whereas the same dose of furosemide did not affect this metabolic pathway.Parallel to these changes, mersalyl caused a significant inhibition in the maximum activity of the most important gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase and glucose 6-phosphatase, in both fed and fasted rats. Neither ethacrynic acid nor furosemide produced any variations in the activities of these enzymes. The activity of the glycolytic enzymes phosphofructokinase and pyruvate kinase was not modified by these diuretics. Nevertheless, the activity of the thiol-enzyme glyceraldehyde 3-phosphate dehydrogenase was severely inhibited by mersalyl and to a lesser extent by the other diuretics. This inhibition was higher in fasted than fed rats. Hence, we conclude that the inhibitory effect of mersalyl on renal gluconeogenesis is due, at least partly, to a decrease in the flux through the gluconeogenic enzymes. Blood glucose was not modified after diuretic treatment in fed animals whereas mersalyl decreased the levels of blood glucose in 24-h fasted rats. Thein vivo effects of diuretics on gluconeogenesis correlate well with the previously observedin vitro effects, although ethacrynic acid was less potent as an inhibitorin vivo, probably because of its rapid clearance.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis (-aminoethylether) N,N,N,N-tetraacetic acid - DTT dithiothreitol - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - TRIS 2-amino-2-hydroxymethyl-1,3-propanediol Publication No. 166 from Drogas, Tóxicos Ambientales y Metabolismo Celular Research Group, Department of Biochemistry and Molecular Biology, University of Granada, Granada, Spain     

Dystrophin predominantly localizes to the transverse tubule/Z-line regions of single ventricular myocytes and exhibits distinct associations with the membrane

Dystrophin is a high molecular weight protein present at low abundance in skeletal, cardiac and smooth muscle and in trace amounts in brain. In skeletal muscle, dystrophin is uniformly distributed along the inner surface of the plasma membrane. Biochemical fractionation studies have shown that all detectable skeletal muscle dystrophin is tightly associated with a complex of wheat germ agglutinin (WGA)-binding and concanavalin A (Con A) binding sarcolemmal glycoproteins. Absence of dystrophin is the primary biochemical defect in patients with Duchenne muscular dystrophy and leads to segmental necrosis of their skeletal myofibers. Although present in similar amounts in normal cardiac and skeletal muscle, the absence of dystrophin from cardiac muscle has less severe effects on the survival of cardiac cells. We have therefore examined whether there are differences in the properties of cardiac and skeletal dystrophin. We report that in contrast to skeletal muscle, cardiac dystrophin is distributed between distinct pools: a soluble cytoplasmic pool, a membrane-bound pool not associated with WGA-binding glycoproteins and a membrane-bound pool associated with WGA-binding glycoproteins. Cardiac dystrophin was not associated with any Con A binding glycoproteins. Immunohistochemical localization studies in isolated ventricular myocytes reveal a distinct punctate staining pattern for dystrophin, approximating to the level of the transverse tubule/Z-line and contrasting with the uniform sarcolemmal staining reported for skeletal muscle fibers. The distinct properties of cardiac dystrophin suggest unique roles for this protein in cardiac versus skeletal muscle function.Abbreviations Dys Dystrophin - T-tubule Transverse tubule - SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - WGA Wheat Germ Agglutinin - Con A Concanavalin A - DHP Dihydropyridine receptor - FITC Fluorescein Isothiocyanate Conjugate - NAG N-Acetyl-D-Glucosamine - NP-40 NONIDET P-40 - PBS Phosphate-Buffered Saline - TBST Tris Buffered Saline-Tween     

Effects of anions, acetazolamide, thiocyanate and amiloride on fluid secretion by the Malpighian tubules of Locusta migratoria L.

The effect of Cl⁻, HCO₃⁻, Br⁻, acetazolamide, thiocyanate and amiloride on urine formation in Locusta migratoria Malpighian tubules have been determined. The rate of fluid secretion depends markedly on the concentration of Cl⁻ in the bathing solution with concentrations less than 90 mM resulting in reduced fluid secretion. Substitution of Br⁻ for Cl⁻ had no significant effect on the rate of the fluid secretion. Replacement of NaHCO₃ with NaCl in Hepes buffered Ringer solution reduced the rate of urine production by 23%. Fluid secretions were reduced in the presence of 10⁻⁴–10⁻² M acetazolamide, a carbonic anhydrase inhibitor. The combined effect of acetazolamide in the absence of HCO₃⁻ appears to be additive. A 1 mM concentration of thiocyanate, an ionic inhibitor, reduced fluid secretion by 35%. Amiloride interferes with the electrogenic entry of Na⁺ into the cell and a 1 mM solution reduced fluid secretion by 94% with secretion completely inhibited in 80% of the tubules tested.