Satellite DNA from the brine shrimp Artemia affects the expression of a flanking gene in yeast.

We have previously revealed that in the brine shrimp Artemia franciscana an AluI DNA family of repeats, 113 bp in length, is the major component of the constitutive heterochromatin and that this repetitive DNA shows a stable curvature that confers a solenoidal geometry on the double helix in vitro. It was suggested that this particular structure may play a relevant role in determining the condensation of the heterochromatin. In this report we have cloned hexamers of highly-repetitive sequence (AluI-satellite DNA) in proximity to a yeast lacZ reporter gene on a plasmid. We find that the expression of the reporter gene is affected by the presence of this DNA in a dose- and orientation-dependent manner in the yeast, S. cerevisiae. We show that this effect is not dependent on under-replication or re-arrangements of the repetitive DNA in the cell but is due to decreased expression of the reporter gene. Our results indicate that the AluI-satellite DNA of Artemia per se is able to influence gene expression.

Apoptosis of hair follicle cells during doxorubicin-induced alopecia in rats.

Alopecia is a common side effect of cancer chemotherapy, and to date, little progress has been made in alopecia prevention or treatment. The present studies were undertaken to topographically localize the site of injury in the hair follicle after doxorubicin (DXR) administration and to investigate the mechanism of DXR-induced alopecia. Tissue samples of head and proximal neck skin obtained from newborn rats treated with DXR were histologically examined by light and electron microscopy and stained by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method. Light microscopy revealed pyknotic cells in the matrix and in the upper bulb and a decrease in the number of mitotic cells. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling analysis evidenced cells with nuclear staining indicative of apoptosis, as confirmed by ultrastructural analysis. Kinetics studies indicated that even a single DXR treatment induced apoptosis and a decrease in mitotically active cells. Our data show that DXR treatment induces injury in a cell subset localized in the hair matrix. The successful prevention of drug-induced alopecia in patients may depend on the selective protection of these cells of the hair follicle.

Anatomical organization of the spinal paths to the soleus and gastrocnemius muscles of the rat hind limb.

The anatomical organization of the motoneuronal columns of the soleus and lateral gastrocnemius muscle and of the related premotor interneurons was studied in rats, using the retrograde transneuronal transport of WGA-HRP. Motoneurons of the gastrocnemius muscle have a well-developed dendritic arborization which spreads into the transverse plane of the spinal cord extending to the intermediate region of the grey matter, while dendrites of the soleus muscle motoneurons spread mainly in the rosto-caudal plane, where they remain inside the border of the motoneuronal column and form small dendritic bundles, suggesting a coupling of neuronal activity as is to be expected in the motoneurons of a tonically active postural muscle such as the soleus. Gastrocnemial premotor interneurons are located close to the motoneuronal column, while the soleus premotor interneurons are scattered all over the ventral horn and intermediate grey. The number of labelled premotor interneurons is greater when the WGA-HRP is injected into the soleus muscle. In both cases, the premotor interneurons could be classified as four different types on the basis of the shape and size of the neuronal somata. The differences in the anatomical organization of the spinal paths to the gastrocnemius and soleus muscles reflect the different tasks performed by these two synergic muscles in normal motor behaviour: fast phasic activity by the gastrocnemius muscle, slow tonic anti-gravity activity by the soleus muscle.

Ultrastructural aspects of DRG satellite cell involvement in experimental cisplatin neuronopathy.

Different substances may induce neurological impairment, clinically expressed as peripheral neuropathies, due to damage of the neuronal bodies (neuronopathy) of sensory or motor neurons. Neuronopathies have generally been studied referring to neurons, although other cellular components may also be damaged. Cisplatin (CDDP) is known to be neurotoxic to the neurons of the dorsal root ganglia (DRG). The scarcity of information as to the possible involvement and role played by dorsal root ganglion (DRG) satellite cells in neuronopathies prompted this study using the chronic DRG neuronopathy induced by the repeated administration of CDDP in rats as a model. Eighteen female Wistar rats were treated according to 3 different schedules of CDDP administration (6 rats for each group). Six further animals were used as controls. At the end of the experiment the L4-L5-L6 dorsal root ganglia were examined at the light and electron microscope. Ag-NOR reaction was also examined in 4 further CDDP-treated rats and 4 controls. Pathological changes in satellite cells of animals treated with CDDP were remarkable in the nucleus where heterochromatin clumps were reduced or even completely absent. Morphometric analysis of the area occupied by heterochromatin indicated that this nuclear component decreased in an exposure-time dependent manner. Frequently, nucleolar-like structures became apparent in the nucleus of the rats treated with the higher doses of CDDP. Ag-NOR positive regions in the nuclei of treated rats were increased with respect to the controls. Cytoplasmic changes in DRG satellite cells of CDDP treated rats were limited, being characterized by an increased electron-density of the matrix. In treated rats deep invaginations between satellite cells and the neuronal surface were evident, leading to the formation of vacuoli. The interstitial connective space often showed edematous areas. Our observations demonstrate that in chronic cisplatin neuronopathy, DRG satellite cells are also involved in the pathological changes induced by drug exposure, and that these changes may be interpreted as being mainly reactive.

An ultrastructural study of neuronal changes in dorsal root ganglia (DRG) of rats after chronic cisplatin administrations.

In humans, the main dose-limiting side-effect of cisplatin (CDDP) treatment is a peripheral sensory neuropathy secondary to dorsal root ganglion (DRG) neuron involvement. To investigate further for neuronal alterations responsible for CDDP neurotoxicity we undertook the present experimental ultrastructural study, based on observations of 3 different groups of rats (6 animals in each group). Group A rats were treated with 1 mg/kg weekly for 9 weeks; Group B with 2 mg/kg weekly for 9 weeks; and group C rats served as untreated controls. At the end of the experiment, rats were perfused with 3% glutaraldehyde and lumbar DRGs were prepared for electron microscopic observations. In CDDP-treated rats somatic, nuclear and, above all, nucleolar size was reduced. Ultrastructurally, the nucleolus was the most affected structure. Nucleolar alterations were quantified morphometrically. Less marked changes were seen in the nucleus and in the RER and Golgi apparatus of the cytoplasm. The number of lysosomes and lipofuscins was greatly increased in CDDP-treated rats. The ultrastructural alterations observed in CDDP rats suggest that CDDP may be neurotoxic due to a reduction in protein synthesis. This assumption would explain why cells such as neurons, which are non replicating, but which have a high rate of protein synthesis, may be the target of the neurotoxic action of CDDP. The lack of an efficient blood/nerve barrier in the DRG explains the involvement of this particular type of neuron.

Randomized, prospective trial of noninvasive positive pressure ventilation in acute respiratory failure.

Recent studies suggest that noninvasive positive pressure ventilation (NPPV) administered by nasal or oronasal mask avoids the need for endotracheal intubation, rapidly improves vital signs, gas exchange, and sense of dyspnea, and may reduce mortality in selected patients with acute respiratory failure, but few controlled trials have been done. The present study used a randomized prospective design to evaluate the possible benefits of NPPV plus standard therapy versus standard therapy alone in patients with acute respiratory failure. Patients to receive NPPV were comfortably fitted with a standard nasal mask connected to a BiPAP ventilatory assist device (Respironics, Inc., Murrysville, PA) in the patient flow-triggered/time-triggered (S/T) mode, and standard therapy consisted of all other treatments deemed necessary by the primary physician, including endotracheal intubation. The need for intubation was reduced from 73% in the standard therapy group (11 of 15 patients) to 31% in the NPPV group (5 of 16 patients, p < 0.05). Among chronic obstructive pulmonary disease (COPD) patients, the reduction was even more striking, with 8 of 12 (67%) control patients requiring intubation compared with 1 of 11 (9%) NPPV patients (p < 0.05). Heart and respiratory rates were significantly lower in the NPPV group than in control patients within 1 h, and PaO2 was significantly improved in the NPPV group for the first 6 h. Dyspnea scores and maximal inspiratory pressures were better in the NPPV than in control patients at 6 h, and nurses and therapists spent similar amounts of time at the bedside for both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Cisplatin induces apoptosis in SH-SY5Y human neuroblastoma cell line.

BACKGROUND: Cisplatin (CDDP) is cytotoxic, inducing apoptosis in some tumoral cell lines in vitro. Since CDDP is an effective drug in vivo treatment of neuroblastoma, we tested this drug on the human neuroblastoma SH-SY5Y cell line. Materials and methods. The effect of CDDP on cultured SH-SY5Y cells was determined with trypan blue dye exclusion test, LDH activity and DNA electrophoresis. Cultures were observed by light and electron microscope. Flow cytometric analysis was also carried out. RESULTS: CDDP inhibits the growth of neuroblastoma cells and reduces cell viability. Cell death occurs by apoptosis, as evidenced by morphological criteria and typical DNA laddering. Flow cytometry demonstrates that CDDP-treated cells are arrested in the G2/M phase before entering programmed cell death. CONCLUSIONS: CDDP is thus effective on the human neuroblastoma SH-SY5Y cell line, inducing apoptosis.

Ultrastructural and confocal laser scanning microscopical aspects of apoptosis in cultured human neuroblastoma SH-SY5Y cells.

Programmed cell death or apoptosis is a physiological process that plays an important role during development and maintains tissue homeostasis during adult life. In pathological conditions, such as cancer, apoptosis may be the mechanism by which cancer proliferation is hampered. Many antineoplastic drugs act by inducing apoptosis. SH-SY5Y human neuroblastoma cells undergo apoptosis when exposed to cisplatin, an effective antineoplastic drug. The occurrence of cell death by the apoptotic process is evidenced by the typical electrophoretic laddering of DNA, which begins 24 h after cisplatin exposure and becomes even more apparent at 3-4 days after exposure. Concomitantly, ultrastructural changes of the nucleus and nucleolar organization occur, followed by nuclear membrane disruption and, finally, by cytoplasm degeneration. These last two aspects are present in cultured cells detached from the substrate and predominate in long-term cultures after drug exposure. Confocal laser scanning microscopy (CLSM) of orange-acridine stained nuclei also clearly demonstrates the fragmentation of the chromatin into 3-5 domains. The CLSM, therefore can clearly demonstrate the occurrence of apoptosis in a much simpler, but equally accurate way than electron microscopy.