Biosynthesis of globin chains in fetal liver and adult marrow cultures : Comparative analysis of individual colonies derived from early, intermediate or late erythroid progenitors

The relative synthesis of globin chains (α,β,^Gγ,^Aγ) has been comparatively evaluated in erythroid colonies from 26 fetal livers (7–15 gestational week) and 13 ‘normal’ adult marrows. Clusters deriving from erythroid colony-forming units (CFU-E) were analysed either individually or in pools of –20 colonies. Bursts deriving from earlier erythroid progenitors (erythroid burst-forming unit, ‘primitive’ or ‘mature’, P-BFU-E or M-BFU-E, respectively) were always analysed individually. Since γ-globin synthesis peaks earlier than β-chain production in both the fetal and the adult erythroblastic pathway, the globin synthetic pattern has been comparatively evaluated, in so far as possible, in colonies at an homogenous, advanced stage of hemoglobinization.In fetal liver cultures, the relative β-synthesis in CFU-E clusters, M- and P-BFU-E bursts constantly shows low, fairly uniform values. In adult marrow cultures, the relative γ-production in the corresponding three classes of colonies is characterized by low, rather homogeneous levels (except for more elevated γ-synthetic values occasionally observed in pooled CFU-E clusters comprising a majority of poorly-hemoglobinized colonies). A gradual decrease of relative γ-production has never been observed in colonies deriving from progressively more differentiated erythroid progenitors of both fetal and adult origin.These results suggest that fetal and adult BFU-E are endowed respectively with a program for prevailing HbF or HbA synthesis, which is not substantially modulated at the level of erythroid progenitors under standard culture conditions. By implication, it is postulated that, in fetal and more particularly adult age, modulation of globin synthesis is mediated via mechanism(s) acting at the level of erythroblasts, i.e. at the level of the early γ- and the late β-synthesis in their maturation pathway. The Hb switch (i.e. the switch from prevailingly HbF to HbA synthesis program) is possibly dependent on the ontogenic ‘maturation’ of BFU-E (and/or stem cells), which peaks in the perinatal period.     

Sulfonamide inhibition profile of the γ-carbonic anhydrase identified in the genome of the pathogenic bacterium Burkholderia pseudomallei the etiological agent responsible of melioidosis

A new γ-carbonic anhydrase (CA, EC 4.1.1.1) was cloned and characterized kinetically in the genome of the bacterial pathogen Burkholderia pseudomallei, the etiological agent of melioidosis, an endemic disease of tropical and sub-tropical regions of the world. The catalytic activity of this new enzyme, BpsCAγ, is significant with a k_cat of 5.3 × 10⁵ s^?1 and k_cat/K_m of 2.5 × 10⁷ M^?1 × s^?1 for the physiologic CO₂ hydration reaction. The inhibition constant value for this enzyme for 39 sulfonamide inhibitors was obtained. Acetazolamide, benzolamide and metanilamide were the most effective (K_Is of 149–653 nM) inhibitors of BpsCAγ activity, whereas other sulfonamides/sulfamates such as ethoxzolamide, topiramate, sulpiride, indisulam, sulthiame and saccharin were active in the micromolar range (K_Is of 1.27–9.56 μM). As Burkholderia pseudomallei is resistant to many classical antibiotics, identifying compounds that interfere with crucial enzymes in the B. pseudomallei life cycle may lead to antibiotics with novel mechanisms of action.     

The pattern of cell division in the early development of the sea urchin,Paracentrotus lividus

In the sea urchin Paracentrotus lividus, the first three cleavages are synchronous in all blastomeres. Upon the segregation of the micromeres at the fourth cleavage, a vegetal-animal gradient of cell division begins; i.e., the closer the cells are to the micromeres, the earlier they enter mitosis. The phase difference between mitotic cells along the vegetal-animal axis increases concurrently with the increase in cell number. At the blastula stage, mitoses appear organized in clusters. From the mesenchyme-blastula stage onward, the clusters become smaller and most of the mitotic cells are solitary. The size of the clusters increases upon treatment with colchicine, thus suggesting the existence of pacemaker units of mitotic activity. We confirm that the division of the micromeres is out of phase with respect to the other blastomeres. Of the eight cells originating from the first division of the micromeres, only the four outer ones continue to cleave; the four inner ones appear to have lost the ability to divide. The overall mitotic activity is high during cleavage and suddenly drops to very low levels around hatching. An interpretation of these results is presented in terms of a nonlinear oscillators theory.     

Suspension culture reveals a morphogenetic property of a thyroid epithelial cell line

It is known that freshly dissociated thyroid cell clusters form follicles in suspension culture. Thyroid epithelial cell lines, grown for many generations in vitro, fail to show colloid-containing lumina when cultured as monolayers. Several thyroid cell lines, some transformed, have been tested with respect to their ability to form extracellular lumina when transferred from monolayer to suspension culture. One cell line in particular, the T78 cell line, showed this property when cultured in suspension. Lumina formed within 3 days even in the absence of added thyrotropin (TSH). The ultrastructure of lumina within cell aggregates resembled that of the thyroid follicle in vivo. The ability to undergo morphogenesis may therefore be an intrinsic property of thyroid epithelial cells which is retained for a large number of generations in vitro and is revealed by proper culture conditions. The shift from monolayer to suspension culture may thus lead to the expression of a thyroid differentiated function such as the formation of follicle-like structures.     

Evaluation of the mixed interaction between apolipoproteins A-II and C-I by equilibrium sedimentation

The mixed interaction between human apolipoproteins C-I and A-II, each of which self-associate in aqueous solution, has been evaluated by sedimentation equilibrium measurements. In order to simplify data analysis apoC-I and apoA-II were modified by treatment with 2-nitrophenylsulfenyl chloride and tetranitromethane respectively. The molecular properties of the resulting derivatives, S-apoC-I and N-apoA-II, each of which have appreciable extinction coefficients above 350 run, were indistinguishable from the corresponding unmodified species. Sedimentation equilibrium data were obtained with mixtures of S-apoC-I and native apoA-II, N-apoA-II and native apoC-I, and native apoC-I and native apoA-II. Mixed complex formation was detected readily with all mixtures investigated. The combined results were most consistent with a single mixed oligomer containing 2 molecules of apoA-II and 4 molecules of apoC-I. The corresponding equilibrium constant was 31 248 ± 890 ($\text{XXX}\text{gm}$)⁵.     

Interaction between bacterial enteric pathogens and aquatic macrophytes. Can Salmonella be internalized in the plants used in phytoremediation processes?

Abstract

The environment is considered a reservoir of pathogens and a possible source of infection for animals and humans. The association between enteric pathogens and food plants has been demonstrated in several studies, while few studies have addressed possible interactions between human pathogens and aquatic plants. This study, performed by setting mesocosms, evaluates the interaction between an enteric pathogen (Salmonella enterica serovar Napoli, S. Napoli) and a macrophyte (Phragmites australis (Cav.) Trin. ex Steudel) and the possible ability of the bacterium to internalize into the plant. The results show that S. Napoli concentration decreased gradually in growth solution without plants (control) while it was able to persist adhering to submerged parts of plants in treated mesocosms. The adhesion of the bacterium remained stable for 20?days, then decreased gradually until the end of the experiment. In addition, S. Napoli was able to internalize and colonize stems and leaves. In conclusion, the study suggests that macrophytes can represent an alternative environmental reservoir of pathogens for humans and animals. The adhesion to roots and rhizomes and the internalization could contribute to the bacterial persistence in the aquatic ecosystems by playing an important role in ecology and transmission of pathogens.     

Biosynthesis of Hb in individual fetal liver bursts : γ-Chain production peaks earlier than β-chain in the erythropoietic pathway

The relative synthesis of α-, β-, ^Gγ- and ^Aγ-globin chains has been evaluated in single fetal liver bursts, which were grown in methylcellulose cultures, individually labelled with [³H]leucine and then analysed via iso-electric focusing. Well-hemoglobinized bursts demonstrate a homogeneous globin synthetic pattern, characterized by prevalent HbF (+some HbA) synthesis: thus, they apparently originate from a homogeneously programmed population of erythroid burst-forming unit (BFU-E). On day 8–9 of culture, the synthetic pattern in ‘mature’ (i.e., well-hemoglobinized) bursts has been compared with that in simultaneously-grown, ‘immature’ (i.e., poorly-hemoglobinized) colonies. These patterns have been further compared with that in ‘matured’ bursts (identified in situ as immature on day 8–9 and labelled 2–4 days later when matured). The ‘immature’ colonies showed very low levels of relative β-globin synthesis, while the ‘mature’ ones demonstrated a more elevated production of β-chain. Significantly, the ‘matured’ bursts showed a globin chain synthetic pattern similar to that of previously labelled ‘matured’ colonies. It is postulated therefore that in fetal liver (and also in adult marrow) the synthesis of γ-chain is linked to an early differentiation stage of erythroblasts, while β-globin synthesis is largely activated at a more advanced maturation stage.