Cellular mechanisms of intestine regeneration in the sea cucumber, Holothuria glaberrima Selenka (Holothuroidea:Echinodermata).

Echinoderms are the deuterostome group with the most striking capacity to regenerate lost body parts. In particular, members of the class Holothuroidea are able to regenerate most of their internal organs following a typical evisceration process. Such formation of new viscera in an adult organism provides a unique model to study the process of organogenesis. We have studied this process in the sea cucumber Holothuria glabberrima by describing the spatial and temporal pattern of cellular events that occur during intestine regeneration following chemically induced evisceration. Regeneration begins as a thickening of the mesenteries that supported the autotomized organs to the body wall. The mesenterial thickening consists of tissues where most of the cellular populations found in the normal intestine are already present. However, the cell numbers differ, particularly those of hemocytes and amoebocytes, suggesting that some of these cells play an important role in the formation of the solid rod of hypertrophic mesentery that characterizes the intestinal primordia. The appearance of the luminal epithelium, together with the formation of the lumen, occurs during the second week of regeneration by proliferation and extensive migration of cells from the esophagus and cloacal ends into the thickenings. At this stage all tissue layers are present, but it takes an additional week for them to exhibit the proportions typical of the normal organ. Cell division, as determined by BrdU labeling, mainly occurs in the coelomic epithelia of the hypertrophic mesentery and in the regenerating luminal epithelium. Our study provides evidence that the process of new organ formation in holothurians can be described as an intermediate process showing characteristics of both epimorphic and morphallactic phenomena.

Substrate selectivity of mouse N-acetyltransferases 1, 2, and 3 expressed in COS-1 cells.

Two human acetyl-CoA:arylamine N-acetyltransferases (NAT1 and NAT2) have been identified. Therapeutic and carcinogenic agents that are substrates for these isoenzymes (including isoniazid, sulfamethazine, p-aminobenzoic acid, 5-aminosalicyclic acid, and 2-aminofluorene) have been used to evaluate the role of the N-acetylation polymorphisms of NAT1 and NAT2 in the treatment of disease and differential risk of various cancers among individuals of differing acetylator phenotypes. The mouse is frequently used as a model of the human acetylator polymorphism. As three Nat isoenzymes have been identified in mouse, it is necessary to determine the selectivity of mouse Nats toward common NAT substrates. In the present study, Nat1*, Nat2*8, and Nat3* were expressed in COS-1 cells, and their substrate selectivity was evaluated with various substrates. Under the conditions used, mouse Nat2 had 20-, 2.4-, and 5.4-fold higher catalytic activity for p-aminobenzoic acid, 5-aminosalicylic acid, and 2-aminofluorene, respectively, than Nat1. Isoniazid N-acetylation was catalyzed only by mouse Nat1. For the substrates tested in this study, mouse Nat3 exhibited activity only toward 5-aminosalicylic acid and only at 1/20 the activity shown by Nat2. In addition, p-aminobenzoylglutamate, the first endogenous NAT substrate identified, was selective for mouse Nat2. These results further support the functional analogy of mouse Nat2 and human NAT1.

Characterization of a hormone response element in the mouse N-acetyltransferase 2 (Nat2*) promoter.

Multiple variant alleles of the human arylamine N-acetyltransferase genes, NAT1* and NAT2*, alter the capacity of individuals to metabolize arylamines by N-acetylation. Although biochemical and genetic studies have improved our understanding of the molecular basis of the acetylation polymorphism in humans and other mammals, regulation of NAT* gene expression is not understood. In the present study, a segment of the 5'-untranslated region of mouse Nat2* was sequenced and characterized. Primer extension analysis and RNase protection assays exposed multiple transcription initiation sites located 112 to 151 bases upstream of the translational start site. Computer sequence analysis revealed a promoter-like region located within the region 530 bases upstream of the translational start site consisting of TATA boxes, upstream promoter elements such as a CAAT box and Sp1 binding site, regulatory elements such as a palindromic hormone response element (HRE), and enhancer regions such as an AP-1 transcription factor binding site. Transient expression of CAT reporter constructs of the mouse Nat2*-palindromic HRE demonstrated positive regulation of the HSV-thymidine kinase 1 (tk1) promoter and induced the expression of chloramphenicol acetyltransferase (CAT). This induction was initiated by the addition of hormones such as 5alpha-dihydrotestosterone (DHT) or dexamethasone and was entirely dependent on the presence of androgen or glucocorticoid receptors, respectively. Together with recent discoveries regarding the effects of testosterone on the expression of Nat2* in mouse kidney during development, the findings reported in this article suggest that the HRE found in the promoter region of Nat2* is a potential candidate for the mediation of androgenic regulation of Nat2* in mouse kidney.