Flow cytometric evaluation of proliferative activity and ploidy in myelodysplastic syndromes and acute leukemias

Propidium iodide (PI) DNA distribution of bone marrow (BM) cells was studied by flow cytometry (FCM) in 36 patients without hematologic or malignant disease (normal BM) and in 172 patients with anemias (36 pts), myelodysplastic syndromes (MDS) (33 pts) and acute leukemia (AL) at diagnosis (60 pts), remission (24 pts) and relapse (19 pts). White blood cells from normal male subjects were used as an external diploid reference standard (median CV = 3.8). Patients with normal BM, anemias, MDS and acute leukemia at diagnosis had tritiated thymidine labeling index (LI) and most with MDS and AL had also evaluable cytogenetics performed on the same BM sample used for FCM. In normal BM, median aliquot of cells with PI-DNA content intermediate between the diploid and the tetraploid value (2n-4n cells %) was 15.7. The ratio between the fluorescence intensity of the G0/1 peak of normal BM cells and the fluorescence intensity of the G0/1 peak of the reference standard (FI ratio) ranged from 93 to 1.05 (mean +/- 2SD). The 2n-4n cell % was higher than normal in anemias (p less than .001), lower in leukemias (p less than .001) and widely scattered in MDS. A linear correlation was found between 2n-4n cell % and LI, with 2n-4n cell % value higher than LI. The FI ratio was lower than normal in anemias (p less than .05), higher in AL with normal cytogenetics (p less than .02) and broadly scattered in MDS with normal cytogenetics. From our experience, PI-DNA-FCM is a simple and adequate method to evaluate proliferative activity in hematologic diseases. Nevertheless, caution must be taken in attributing small changes in FI ratio to aneuploidy, since they are found in anemias and in MDS and AL with normal cytogenetics, possibly due to differences in PI uptake by different cell types.     

Spermatophore development and sperm ultrastructure inCraterostigmus tasmanianus (Chilopoda,Craterostigmomorpha)

 Spermatophore development and ultrastructure of the mature sperm of Craterostigmus tasmanianus were studied using light and electron microscopy. In C. tasmanianus, as in the Scolopendromorpha, the spermatophore develops within the vas deferens. The latter consists of three parts, each with a different morphology. The first may be involved in guiding the sperm to roll up into typical ring-like structures, while the other two, which show an evident secretory activity, secrete the acellular wall of the spermatophores. The ultrastructure of mature spermatozoa showed that a very close similarity exists between Craterostigmomorpha and Lithobiomorpha, especially regarding the organization of the connecting piece. Based on this similarity, we consider the Craterostigmomorpha together with the Scolopendromorpha, Geophilomorpha and Lithobiomorpha (=Pleurostigmophora) to be the sister group of the Scutigeromorpha. Accepted: 2 June 1996     

Unusual conformational preferences of β-alanine containing cyclic peptides. VII

In the present paper we describe the synthesis, purification, and single crystal x-ray analysis of the cyclic pentapeptide cyclo-(Pro-Phe-Phe-β-Ala-β-Ala). This compound crystallizes in the orthorhombic space group P2₁2₁2₁ from methanol and adopts in the solid state an unusual conformation characterized by a cis β-Ala⁵-Pro¹ peptide bond and by an intramolecular hydrogen bond stabilizing a C₁₁- and a C₁₂-ring structure. The C₁₁, structure contains the Phe³ and the β-Ala⁴ at the corner position of the turn; it is the first observation of a type II β-turn enlargement due to the insertion of an extra methylene group of the β-alanine residue. The rest of the molecule participates in a newly characterized C₁₂-ring structure, which incorporates a β-Ala residue at position i of the turn. © 1996 John Wiley & Sons, Inc.     

Normal rat intestinal cells IEC-18: characterization and transfection with immortalizing oncogenes

IEC-18 cells, a cell line derived from the ileum of rat intestine, have the characteristics of normal cells since they have a contact inhibited cell growth, do not form colonies in soft agar and are not tumorigenic when injected in nude mice. IEC-18 cells were transfected with nuclear oncogenes, c-myc, v-myc and SV40 T antigen in order to obtain immortal cell lines. Independent clones were isolated and characterized for the growth properties. Expression of v-myc altered the morphology of the cells and shortened the doubling time. A slow growth together with a low cloning efficiency was associated with the expression of SV40 T antigen. No changes either in growth or in morphology were observed in c-myc-expressing IEC-18 cells. Expression of these nuclear oncogenes did not result in the neoplastic transformation of the IEC-18 cells, since none of the clones lost the anchorage dependence or were able to form tumors in vivo. The c-myc-containing IEC-18 cells were unable to secrete in the growth medium TGF and exposure to TGF inhibited the growth rate by 30%. All these observations are consistent with the conclusion that the expression of nuclear oncogenes does not lead to the neoplastic transformation of these cells.     

An oxygenation-sensitive dye binding to Carcinus maenas hemocyanin

The interaction of 4',6-diamidino-2-phenylindole (DAPI) with Carcinus maenas hemocyanin has been investigated by steady state fluorescence, dynamic fluorescence and circular dichroism measurements. The dye binds to apohemocyanin (without copper) as well as to oxygenated hemocyanin and to deoxygenated hemocyanin with very similar affinities (kd approximately equal to 1 microM ) and number of binding sites (one per subunit). In contrast, the fluorescence quantum yield enhancement of DAPI bound to oxygenated hemocyanin is nearly 60% lower than that observed for deoxygenated and apo forms. The decrease of fluorescence of the dye bound to deoxygenated hemocyanin is a sigmoidal function of the oxygen partial pressure, specular to that observed by following the absorbance of the copper-oxygen charge transfer band at 340 nm. This result provides preliminary evidence that DAPI may be used as a functional probe to monitor the cooperative binding of oxygen to the protein. The higher fluorescence quantum yield of DAPI bound to either apohemocyanin or deoxygenated protein is characterized by a single fluorescence decay with lifetime of about 3 ns, while with the oxygenated protein two components of about 1 ns and 3.0 ns are observed. This result is interpreted assuming the existence of two rotamers of DAPI in solution (Szabo et al. Photochem. Photobiol. 44 (1986) 143-150) both able to interact with oxygenated hemocyanin but only one to deoxygenated and apo forms. We conclude that the different fluorescence behaviour of the dye induced by the presence of oxygen bound to the protein is probably due to a structural change of hemocyanin in cooperative interaction with oxygen. Furthermore, the interaction is confirmed by the induced negative ellipticity of DAPI bound to apohemocyanin and deoxy-hemocyanin and by the increase of fluorescence anisotropy of DAPI bound to all forms of protein investigated.     

An Ultrastructural Investigation on Vitellophage Invasion of the Yolk Mass during and after Germ Band Formation in Embryos of the Stick Insect Carausius morosus Br.

Developing embryos of the stick insect Carausius morosus were examined ultrastructurally with a view to studying vitellophage invasion of the yolk mass during and after germ band formation. Newly laid eggs in C.morosus have a unique yolk fluid compartment surrounded by a narrow fringe of cytoplasm comprising several small yolk granules. Vitellophages originate mainly from a thin layer of stem cells, the so-called yolk cell membrane, interposed between the germ band and the yolk mass. Throughout development, a thin basal lamina separates the yolk cell membrane from the overlying embryo.
Vitellophages extend from the yolk cell membrane with long cytoplasmic processes or filopodia to invade the central yolk mass. Along their route of entrance, filopodia engulf portions of the yolk mass and sequester it into membrane-bounded granules. As this process continues, the yolk mass is gradually partitioned into a number of yolk granules inside the vitellophages.
Later in development, the yolk cell membrane is gradually replaced by the endodermal cells that emerge from the anterior and posterior embryonic rudiments. From this stage of development onwards, vitellophages remain attached to the basal lamina through long filopodia extending between the endodermal cells. Yolk confined in different vitellophagic cells appears heterogeneous both in density and texture, suggesting that yolk degradation may be spatially differentiated.     

Melatonin controls cell proliferation and modulates mitochondrial physiology in pancreatic stellate cells

Journal of Physiology and Biochemistry - We have investigated the effects of melatonin on major pathways related with cellular proliferation and energetic metabolism in pancreatic stellate cells....     

Combined effect of ambient temperature and solar radiation on maned sloths' behaviour and detectability

Changes in ambient temperature and solar radiation may affect sloths' metabolic rate and body temperature, with consequent changes in activities, postures and microhabitat selection. Although the separate effect of temperature and solar radiation on sloth's behaviour have been previously studied, the combined effect of these climatic factors on behavioural aspects of sloths has never been systematically evaluated in field conditions. Here we evaluated the influence of hourly ambient temperature variation on maned sloth (Bradypus torquatus) activities, postures and tree crown positions, under sunny and cloudy conditions; and tested if any of the animal posture and position increase their exposure to human detection. We performed 350 h of visual observation on eight maned sloths, equipped with radio-backpacks, in northern Bahia, Brazil, recording their activities, and their resting postures and positions on tree crowns. We also recorded the time taken to visualize the sloths on 58 days to analyse if sloths' detection is affected by posture and position. Higher ambient temperature, within a range of 21–33°C, increased the sloths' activity levels in cloudy conditions but reduced their activity in sunny conditions. Increasing ambient temperature also reduced the frequency of huddled posture and increased the frequency of extended posture and permanence in the inner tree crown. Lastly, the postures and positions did not influence sloths' detectability. Thus, the direction of the temperature–activity relationship depends on climatic conditions (sunny/cloudy), and individuals rely on resting postures and positions to thermoregulate. The warmer and drier future climate, expected to occur in the northern Atlantic Forest, may impose change in the diurnal activity levels and postural pattern for this threatened species, leading maned sloths to reduce its activity on sunny and warmer days and adopting an extended posture.     

The binding of 1,N6-etheno-NAD to bovine liver glutamate dehydrogenase

The interaction of thrombin and platelets was studied with a heterobifunctional photoactivable crosslinking agent. Radiolabeled thrombin that was modified with ethyl-N-5-azido-2-nitrobenzoylaminoacetimidate formed two types of complex with platelet proteins: platelet-associated complexes and supernatant complexes. The platelet-associated complexes formed within 20 s. Autoradiography after electrophoresis with sodium dodecyl sulfate indicated that these complexes had apparent masses of 210, 185, 155 and 125 kDa. Formation of the complexes was blocked by hirudin; this is consistent with crosslinking that was a direct consequence of the binding of thrombin to a specific receptor, since hirudin blocks thrombin-induced platelet activation and the saturable binding of thrombin to platelets. The labeled supernatant complex had an apparent mass of about 490 kDa. It also formed in the supernatant solution of platelets after activation with a divalent cation ionophore, suggesting a complex of thrombin with a secreted protein. The supernatant complex did not involve fibrinogen or alpha 2-macroglobulin, but a similar complex was formed with partially purified secreted glycoprotein G (thrombin-sensitive protein, thrombospondin). Formation of the complex was blocked by hirudin. A similar complex was formed after prolonged (1 h) incubation without photoactivation. It is concluded that thrombin forms high-affinity, hirudin-sensitive complexes with secreted glycoprotein G, as well as with platelet surface proteins.     

MK-801 Prevents 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Parkinsonism in Primates

In cynomologus monkeys, systemic administration of MK-801, a noncompetitive antagonist for the N-methyl-D-aspartate receptor, prevented the development of the parkinsonian syndrome induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MK-801 also attenuated dopamine depletion in the caudate and putamen and protected dopaminergic neurons in the substantia nigra from the degeneration induced by the neurotoxin. Nevertheless, 7 days after MPTP administration in the caudate and putamen of monkeys also receiving MK-801, the levels of toxic 1-methyl-4-phenylpyridinium were even higher than those measured in monkeys receiving MPTP alone. This indicates that the protective action of MK-801 is not related to MPTP metabolism and strongly suggests that, in primates, the excitatory amino acids could play a crucial role in the mechanism of the selective neuronal death induced by MPTP.