Down-regulation of statin,a nonproliferation-specific nuclear protein,and up-regulation of c-myc after initiation of programmed cell death in mouse fibroblasts

Deprivation of growth factors has been shown to induce programmed cell death in many cell types, including mouse 3T3 fibroblasts. Programmed cell death (apoptosis) is an active process of self-destruction which is thought to require the expression of unique genes. Recently, the expression of cell cycle genes such as c-fos and c-myc, and re-entrance to cell cycle traverse, are thought to be necessary to induce programmed cell death. Previous work in this laboratory has shown that statin is a nonproliferation-specific nuclear protein present in the nuclei of young quiescent or senescent human fibroblasts, as well as in growth-arrested mouse 3T3 fibroblasts; we have reported that statin disappears rapidly after the blockage of growth arrest is removed and cells are allowed to resume cell cycle traverse. In this report we address the question of whether cells induced to enter the programmed cell death process also lose the expression of statin. We studied density-arrested quiescent mouse 3T3 cells, which undergo rapid cell death by apoptosis upon serum deprivation. Our results suggest that c-myc expression is induced, as previously reported in other systems of apoptotic death. Interestingly, we also find that statin indeed disappears after the induction of programmed cell death is initiated. These results further support the notion that when apoptosis is induced, cells behave as though released from replication arrest, and experience some part of the G₁ phase of the cell cycle. The difference between this event and normal cell cycle traverse is that this experience of the G₁ phase in the apoptotic process is an abortive one, with the end result of cell demise. © 1995 Wiley-Liss, Inc.     

Product of aromatase activity in intact LNCaP and MCF-7 human cancer cells

We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 μM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (Δ⁴Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 μM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to Δ⁴Ad. In either cell line, T transformation to Δ⁴Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.     

THE ANATOMY OF THE PISTILLATE INFLORESCENCE AND FLOWER OF CASUARINA VERTICILLATA LAMARCK (CASUARINACEAE)

The pistillate inflorescence of Casuarina verticillata is described as consisting of a primary axis bearing whorls of bracts with a cymule in the axil of each bract of the more central whorls. Each cymule consists of an atepallate, two-carpellate, syncarpous floret and two, lateral, once-lobed bracteoles. A “peripheral intercalary” meristem, in which divisions are primarily periclinal, forms a meshwork beneath the bracts from early development and moves the connate bracts centrifugally around the cymules and extends and binds the bracts, and to some extent the bracteoles, of the fertile part of the inflorescence together. Each bract receives a single trace; each cymule receives two traces. Each bundle extension of a cymule trace supplies: 1) a branch which joins its counterpart to become the anterior common carpellary bundle; 2) a second branch which joins its counterpart to become the posterior common carpellary bundle; and 3) a central branch which supplies a lateral bracteole. Within each floret, each common carpellary bundle provides a dorsal carpellary bundle, two ventral carpellary bundles (fertile anterior carpel) or one common ventral bundle (sterile posterior carpel). The ventral bundle-supplies join and form a single placental bundle which lies in the gynoecial septum, and which, in turn, supplies the two ovules in the anterior carpel. Whether the inflorescence is a simple racemose or a condensed cymose type cannot be determined from this species alone. The function of the sclerenchymatous, enclosing bracteoles and connate bracts is discussed.     

ANATOMY AND ASPECTS OF DEVELOPMENT OF THE STAMINATE INFLORESCENCES AND FLORETS OF SEVEN SPECIES OF ALLOCASUARINA (CASUARINACEAE)

The morphology, ontogeny, and vascular anatomy of the staminate inflorescences and florets of seven species of Allocasuarina are described. The generally terminal but open-ended inflorescences occur on monoecious or staminate dioecious trees and consist of whorls of bracts, each subtending a sessile axillary floret. Each floret consists of one terminal stamen with a bilobed, tetrasporangiate anther enclosed typically by cuculliform appendages, commonly considered bracteoles, an inner median pair and an outer lateral pair. The mature stamen is exerted, the anther is basifixed and is extrorsely dehiscent. In early development of a male inflorescence very little internodal elongation occurs and enclosing cataphylls appear. The inflorescence apex is a low dome with a uniseriate tunica and a small group of central corpus cells. Bract primordia are initiated by periclinal divisions of C₁ followed by further divisions of the corpus and anticlinal divisions in the tunica. The bracts are epinastic and become gamophyllous except apically by cell divisions in both sides of each primordium. Stomata are restricted to the axis furrows and the abaxial tips of the bracts. The axillary florets arise in acropetal succession initiated by periclinal divisions in C₁ accompanied by anticlinal divisions in the tunica. The lateral floral appendages are also initiated by C₁ followed by anticlinal divisions in the tunica. They become adnate basally later with the subtending bract. The median sterile appendages are initiated in a manner similar to the initiation of the outer appendages. The stamen is initiated by divisions in the outer layers of the corpus and in the tunica, and then develops first by apical growth followed by intercalary growth. The vascular system of the inflorescence is identical to that of the vegetative stem. Each floret is supplied by a single bundle that has its source in a branch from each of the two traces supplying a bract. Six bundles arise from the floral bundle; four of these terminate in the base of the stamen and two form an amphicribal bundle that supplies the anther. Pollen is binucleate, 3- to 7-porate. The exine is tegillate.     

Interaction of α2-macroglobulin with L-asparaginase

Summary Obvious protection of the catalytic activity of Esch. coli L-asparaginase by ₂-macroglobulin (₂M) was observed under conditions otherwise propitious to the dissociation of the tetrameric molecule into inactive subunits, i.e. very diluted enzyme solutions or the presence of either SDS or urea. The degree of protection depended on enzyme and ₂M concentrations respectively, and on the preincubation time of the ₂M-enzyme mixture prior to substrate addition. The formation of a catalytically active complex between ₂M and L-asparaginase was confirmed by gel filtration on a Sephadex-G column and by polyacrylamide gel electrophoresis. The fact that the migration distance of the active complex corresponded to the migration of ₂M and the absence in that case of a migration band corresponding to the intact molecule suggest that complexing of the enzyme with ₂M prevented its dissociation into subunits and thus its inactivation. Addition of ₂M to the already dissociated enzyme molecule did not restore its catalytic activity.Alpha₂-macroglobulin was shown to have an inhibiting effect on the proteolytic activity of almost all proteases and no effect on their esterolytic activity. Furthermore, it prevents the inhibition of esterolytic activity by some natural compounds^1–5. The effect of ₂M on other types of catalytic activity has not been investigated enough to afford a generalization of the possible role of this macroglobulin in the control of enzyme activity in the body.This paper reports the results of an in vitro study of the effect of ₂M on the catalytic activity of an important amidase, i.e. L-asparaginase (L-asparagine amidohydrolase 3.5.1.1), which in recent years has been used in the treatment of acute lymphocytic leukemia in children^6,7.Abbreviations ₂M ₂-macroglobulin - E enzyme - SDS sodium dodecylsulfate Part of the results were reported at the 10th International Congress of Biochemistry, Hamburg 1976, Abst. p. 377.     

Role of the bacterial and phage recombination systems and of DNA replication in genetic recombination of UV-irradiated phage lambda

Summary In this paper are studied in E. coli K12 the influence of the bacterial Rec and phage Red recombination systems on the rescue of the O ⁺ gene from the prophage by a superinfecting O ^- phage, UV irradiated or not. In the absence of UV irradiation the Red system produces more recombinants that does the Rec system, and its action requires DNA replication. The presence of UV lesions in the DNA facilitates the action of the Rec system, which is more efficient in this instance than the Red system and can act in the absence of DNA replication. In all cases, there is a cooperation between the two generalized recombination systems.     

Endocytic SNAREs are involved in optimal Coxiella burnetii vacuole development

Coxiella burnetii is a Gram‐negative intracellular bacterium. As previously described, both the endocytic and the autophagic pathways contribute to the maturation of Coxiella replicative vacuoles (CRVs). The large CRVs share the properties of both phagolysosomal and autophagolysosomal compartments. Vamp3, Vamp7 and Vamp8 are v‐SNAREs involved in the endocytic pathway which participate mainly in the fusion between endosomes and lysosomes. In the present study we observed that Vamp7 interacts with C. burnetii at different infection times (1 h–48 h p.i.). We have determined that a truncated mutant of Vamp7 (Vamp7 NT) and a siRNA against this SNARE protein affects the optimal development of CRVs, suggesting that Vamp7 mediates fusion events that are required for the biogenesis of CRVs. Indeed, we have observed that overexpression of Vamp7 NT inhibited the heterotypic fusion with lysosomes and the homotypic fusion between individual Coxiella phagosomes and CRVs. Moreover, we have detected in the vacuole membrane, at different infection times, the Vamp7 partners (Vti1a and Vti1b). Interestingly, treatment with chloramphenicol reduced the colocalization between C. burnetii and Vamp7, Vti1a or Vti1b, indicating that the recruitment of these SNAREs proteins is a bacteria‐driven process that favours the CRV biogenesis, likely by facilitating the interaction with the endolysosomal compartment.     

Gibberellin reverses the negative effect of paclobutrazol but not of chlorocholine chloride on the expression of SGs/GAs biosynthesis-related genes and increases the levels of relevant metabolites in Stevia rebaudiana

Plant Cell, Tissue and Organ Culture (PCTOC) - Steviol glycosides (SGs) and gibberellins (GAs) share the same molecular basis. However, the coordination of their respective biosynthetic pathways is...