Detection and characterisation of NAD(P)H-diaphorase activity in Dictyostelium discoideum cells (Protozoa).

In Dictyostelium discoideum (D. discoideum), compounds generating nitric oxide (NO) inhibit its aggregation and differentiation without altering cyclic guanosine monophosphate (cGMP) production. They do it by preventing initiation of cyclic adenosine monophosphate (cAMP) pulses. Furthermore, these compounds stimulate adenosine diphosphate (ADP)-ribosylation of a 41 kDa cytosolic protein and regulate the glyceraldehyde-3-phospate dehydrogenase activity. Yet, although D. discoideum cells produce NO at a relatively constant rate at the onset of their developmental cycle, there is still no evidence of the presence of nitric oxide synthase (NOS) enzymes. In this work, we detect the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in D. discoideum and we characterise it by specific inhibitors and physical-chemical conditions that allegedly distinguish between NOS-related and -unrelated NADPH-d activity.

Macronuclear chromatin structure dynamics in Colpoda inflata (Protista, Ciliophora) resting encystment.

The chromatin structure dynamics of the Colpoda inflata macronucleus have been investigated in relation to its functional condition, concerning chromatin body extrusion regulating activity. Samples of 2- and 25-day-old resting cysts derived from a standard culture, and of 1-year-old resting cysts derived from a senescent culture, were examined by means of histogram analysis performed on acquired optical microscopy images. Three groups of histograms were detected in each sample. Histogram classification, clustering and matching were assessed in order to obtain the mean histogram of each group. Comparative analysis of the mean histogram showed a similarity in the grey level range of 25-day- and 1-year-old cysts, unlike the wider grey level range found in 2-day-old cysts. Moreover, the respective mean histograms of the three cyst samples appeared rather similar in shape. All this implies that macronuclear chromatin structural features of 1-year-old cysts are common to both cyst standard cultures. The evaluation of the acquired images and their respective histograms evidenced a dynamic state of the macronuclear chromatin, appearing differently condensed in relation to the chromatin body extrusion regulating activity of the macronucleus. The coexistence of a chromatin-decondensed macronucleus with a pycnotic extrusion body suggests that chromatin unable to decondense, thus inactive, is extruded. This finding, along with the presence of chromatin structural features common to standard and senescent cyst populations, supports the occurrence of 'rejuvenated' cell lines from 1-year-old encysted senescent cells, a phenomenon which could be a result of accomplished macronuclear renewal.

A study on macronuclear chromatin structure in Colpoda inflata (Protozoa, Ciliophora) by means of image analysis.

Resting encystment is a reversible cytodifferentiation phenomenon regularly taking place during the life cycle of Colpodidae. The encystment involves regulation of the macronuclear DNA content, accomplished via chromatin extrusion and DNA synthesis mechanisms. An analysis of macronuclear chromatin texture was carried out in Colpoda inflata logarithmically growing cells, pre-cystic cells, and increasingly aging resting cysts after Feulgen staining. Morphometric and densitometric parameters as well as Markovian chromatin texture variables were utilised for image analysis. Moreover, univariate and multivariate analyses were applied both to macronuclei and to their chromatin extrusion bodies. The results show that structural variations in chromatin appear under macronuclear functional activity conditions, suggesting a dynamic state of chromatin texture throughout a long-term resting encystment.

[Synthesis of RNA and DNA in isolated nuclei of Colpoda cucullus (Protozoi Ciliati)]. / Sintesi di RNA e DNA nei nuclei isolati di Colpoda cucullus (Protozoi Ciliati).

The ciliated protozoan Colpoda cucullus has been cultivated at 27 degrees C with gentle shaking in a baked lettuce infusion supplemented with Klebsiella suspensions. Under these conditions cells had a mean generation time of about 7 hours and could attain densities up to 20,000/ml and 45,000/ml in the log and stationary phase of growth, respectively. Nuclear preparations obtained from exponentially growing cells by the gum arabic-octanol method showed a satisfactory degree of purity and integrity. They consisted primarily of the large macronuclei attached to which the small micronuclei were sometimes visible. Upon incubation at 27 degrees C in conventional reaction mixtures nuclear preparations actively incorporated 3H-UTP and 3H-dTTP into acid-insoluble material. alpha-amanitin caused a 50% inhibition of RNA synthesis whereas aphidicolin did not affect at all DNA synthesis.