Carbonic anhydrase inhibition blocks skeletogenesis and echinochrome production in Paracentrotus lividus and Heliocidaris tuberculata embryos and larvae

Carbonic anhydrases (CAs) are a family of widely distributed metalloenzymes, involved in diverse physiological processes. These enzymes catalyse the reversible conversion of carbon dioxide to protons and bicarbonate. At least 19 genes encoding for CAs have been identified in the sea urchin genome, with one of these localized to the skeletogenic mesoderm (primary mesenchyme cells, PMCs). We investigated the effects of a specific inhibitor of CA, acetazolamide (AZ), on development of two sea urchin species with contrasting investment in skeleton production, Paracentrotus lividus and Heliocidaris tuberculata, to determine the role of CA on PMC differentiation, skeletogenesis and on non‐skeletogenic mesodermal (NSM) cells. Embryos were cultured in the presence of AZ from the blastula stage prior to skeleton formation and development to the larval stage was monitored. At the dose of 8 mmol/L AZ, 98% and 90% of P. lividus and H. tuberculata embryos lacked skeleton, respectively. Nevertheless, an almost normal PMC differentiation was indicated by the expression of msp130, a PMC‐specific marker. Strikingly, the AZ‐treated embryos also lacked the echinochrome pigment produced by the pigment cells, a subpopulation of NSM cells with immune activities within the larva. Conversely, all ectoderm and endoderm derivatives and other subpopulations of mesoderm developed normally. The inhibitory effects of AZ were completely reversed after removal of the inhibitor from the medium. Our data, together with new information concerning the involvement of CA on skeleton formation, provide evidence for the first time of a possible role of the CAs in larval immune pigment cells.     

Differential response to hepatic differentiation stimuli of amniotic epithelial cells isolated from four regions of the amniotic membrane

Human Amniotic Epithelial Cells (hAEC) isolated from term placenta are a promising source for regenerative medicine. However, it has long been debated whether the hAEC population consists of heterogeneous or homogeneous cells. In a previous study, we investigated the characteristics of hAEC isolated from four different regions of the amniotic membrane finding significant heterogeneity. The aim of this study was to evaluate the hepatic differentiation capability of hAEC isolated from these four regions. Human term placentae were collected after caesarean section and hAEC were isolated from four regions of the amniotic membrane (R1-R4, according to their relative distance from the umbilical cord) and treated in hepatic differentiation conditions for 14 days. hAEC-derived hepatocyte-like cells showed marked differences in the expression of hepatic markers: R4 showed higher levels of Albumin and Hepatocyte Nuclear Factor (HNF) 4α whereas R1 expressed higher Cytochrome P450 enzymes, both at the gene and protein level. These preliminary results suggest that hAEC isolated from R1 and R4 of the amniotic membrane are more prone to hepatic differentiation. Therefore, the use of hAEC from a specific region of the amniotic membrane should be taken into consideration as it could have an impact on the outcome of therapeutic applications.     

Immunohistology of gastrointestinal carcinoid tumors

DAKO quick staining kits were used to immunostainings for Epithelial Membrane Antigen (EMA) Neuron Specific Enolase (NSE) and S-100 protein (S-100) in 18 carcinoid tumors of the gastrointestinal tract classified according to Soga and Tazawa. EMA was always absent in carcinoid tumors, being at the same time present within glandular epithelium. 88% of cases showed positive immunostaining for NSE. S-100 immunostaining showed immunopositive stellate cells present within the tumor especially within type A carcinoids. In addition in one carcinoid tumor thick, strongly S-100 positive bundles were noticed at the periphery of nests of tumor cells. Combined immunohistochemical and ultrastructural studies are needed to elucidate interrelation of neoplastic and neural elements within carcinoid tumorsa.     

The three-dimensional structure of the zona pellucida in growing and atretic ovarian follicles of the mouse

Summary The present study provides further details on the fine-structural three-dimensional architecture of the zona pellucida (ZP) in growing and atretic follicles of mice by use of ruthenium red in combination with the detergents Triton X100 and saponin. These detergents were used for extraction of the soluble fraction of the zonal proteins in an attempt to expose the structural zonal glycoproteins, which in turn can be viewed as minute three-dimensional networks upon transmission- and scanning electron-microscopic examination. By use of these methods, the ZP of growing follicles appeared to be formed by interconnected filaments which also bind to globular structures building up a three-dimensional lattice. In contrast, the ZP of stage I as well as other (II and III) stages of atretic follicles showed a structure characterized by the presence of closely packed granules connected with short filaments to form a close-mesh reticulum. This structural change of the ZP, which in the present study is also associated with the disappearance of gap junctions within the granulosa and cumulus cell population, might represent one of the early events involved in the onset of atresia. These changes, most probably depending on an altered secretory activity of both oocytes and follicle cells, might lead to a degradation of the ZP network structure and to its subsequent increased density (condensation). All these morphodynamic events eventually contribute to a sequestration of the oocyte in the early stage of atresia.     

Transformation of Streptococcus sanguis to intrinsic penicillin resistance

A series of step-level penicillin-resistant derivatives of Streptococcus sanguis V288 (Challis) were obtained through successive genetic transformations. The DNA donor used was a laboratory-derived, penicillin-resistant multistep mutant of the recipient strain. Detection of the penicillin-binding proteins (PBPs) of wild-type and transformants revealed five major PBPs. While it was found that S. sanguis can acquire intrinsic resistance in a stepwise manner and the mechanism was similar to those of some other organisms (changes in penicillin-binding protein affinity and/or in extent of penicillin binding), multiple-PBP changes accompanied a single step-level of resistance. All of the PBPs showed varying degrees of decreased affinity for [3H]benzylpenicillin with increasing penicillin resistance. Of these, the consistent, dramatic and progressive decrease of PBP 4 binding was most notable. After an initial decrease at the first step-level of resistance, PBP 5 was restored to wild-type levels, indicating a possible important role in survival. Genetic linkage of the first two step-levels of resistance was demonstrated by examination of transformation frequencies and by hit-kinetics experiments. A convenient method is described for the quantitative comparison of fluorographs containing PBPs with a wide range of affinities for penicillin.     

Tricyclic Imipramine Modification of the Circadian Rhythms of Hypothalamic Serotonin, its Precursors and Acid Catabolite in Individually Housed Rats

Circadian rhythms of serotonin (5HT), its precursors tryptophan (TP) and 5-hydroxy-tryptophan (5HTP) and its acid catabolite 5-hydroxy-indoleacetic acid (5HIAA), were determined in the hypothalamus of control rats and rats which had been treated continuously with subcutaneous imipramine (10 mg/kg/day) for 2 weeks.

Rats were individually housed and entrained to LD12:12. Controls showed the 5HT and TP peaks in the light and dark periods respectively, as reported in the literature, but no inverted correlation (antiphase) between SHT and 5HIAA rhythms.

Imipramine significantly modified circadian rhythm characteristics: the 5HT acrophase was advanced, that of TP and 5HIAA was delayed. Imipramine also significantly increased hypothalamic SHT and TP concentrations.     

Chemical modification of essential carboxyl group and histidine residue in the plasma-membrane 5'-nucleotidase

An investigation, using specific chemical reagents, of the amino acids involved in the catalytic activity of the purified 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) from bovine liver plasma membranes, was carried out. The enzyme was irreversibly inactivated by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The inhibition kinetics were of the first-order type and decreased partially in the presence of nucleotides and divalent cations. These results indicate for the first time that a carboxyl group is essential for the catalytic process of 5'-nucleotidase. Moreover, chemical modification by diethylpyrocarbonate also produced inactivation of the enzyme and showed a differential spectrum with a peak at 240 nm characteristic of N-carbethoxyhistidine residues. This inactivation was efficiently released upon decarbethoxylation by hydroxylamine only when the extent of inactivation, due to low concentration of diethylpyrocarbonate, was limited. The time-dependent inactivation followed first-order kinetics and nucleotides afforded significant protection against diethylpyrocarbonate modification. The results indicate the involvement of the histidine residue in catalysis.     

Morphological studies of polycystic mouse ovaries induced by dehydroepiandrosterone

Summary Morphological alterations induced by dehydroepiandrosterone (DHA) were studied in polycystic mouse ovaries (PCO). Treated mice showed ovulatory failure and cystic changes; cysts and follicles in various stages of growth and atresia were present although corpora lutea were absent. The levels of testosterone, dihydrotestosterone, 3- and 3-androstanediol, estrone and androstenedione increased, whereas estradiol was not detectable.The ultrastructure of granulosa cells in healthy and atretic follicles was similar to that of control animals, although the membrana granulosa in cysts was reduced to a monolayer of flattened cells. The theca interna of healthy and atretic follicles and ovarian cysts showed ultrastructural signs of abnormal steroidogenic stimulation.No significant differences (0.7<P<0.8) were found between the extensive surface area of gap junctions of healthy follicles of control and DHA-treated animals. On the P-face of granulosa cells of large healthy follicles, meandering strands of tight junctional particles were observed; their average length was significantly longer than those in healthy follicles of control animals (P<0.001). This increase was probably related to the large amounts of androgens present in the treated animals.Theca interna cells possessed small gap junctions; no significant differences (P>0.9) in gap-junction surface area were observed between DHA-treated and control animals. These results suggest that the size of gap junctions is probably unrelated to the steroidogenic activities of theca cells.The following trivial names have been used: Dihydrotestosterone: 5-androstan 17 ol-13 one; 3-androstanediol: 5-androstan 3,17 diol; 3-androstanediol: 5-androstan 3,17 diol