Estimating duration of HIV infection with CD4 cell count and HIV-1 RNA at presentation.
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Adult↗
Biomedical subjects
A Alessandrini
Publications and source records attributed to A Alessandrini.
Early effects of AZT on mitochondrial functions in the absence of mitochondrial DNA depletion in rat myotubes.
Zidovudine (AZT) is a potent inhibitor of human immunodeficiency virus (HIV) replication. In humans, as well as in animal models, long-term treatment with AZT induces a severe myopathy characterised by structural and functional alterations of mitochondria associated with depletion of mitochondrial DNA (mtDNA). In the present work, we compared the effects induced by AZT on mitochondria upon short- or long-term treatments of cultured rat myotubes. Morphological alterations were investigated by electron microscopy, and mtDNA depletion and deletions were analysed by Southern blot. Mitochondrial membrane potential was determined after JC-1 staining by laser-scanning confocal microscopy in whole cells, and by flow cytometry in isolated muscle mitochondria. We found that the early effects of AZT on mitochondrial functions were a marked, yet reversible reduction in mitochondrial membrane potential, in the absence of any effect on mtDNA. The long-term treatment, in addition to mitochondrial membrane potential alterations, induced morphological changes in mitochondria, and a remarkable reduction in the amount of mtDNA, without any significant evidence of mtDNA deletions. In both treatments, a block of the spontaneous contraction of myotubes was observed. To study in more detail the early effects induced by AZT, the ability of the drug to interact with cardiolipin, an important component of internal mitochondrial membrane, was investigated by atomic force microscopy (AFM) in an artificial membrane model system. The results suggest that the primary effects of AZT may be related to a physical interference with the membrane structure leading to a consequent modification of its physical characteristics.
Animals↗
Intravenous administration of MEK inhibitor U0126 affords brain protection against forebrain ischemia and focal cerebral ischemia.
Brain subjected to acute ischemic attack caused by an arterial blockage needs immediate arterial recanalization. However, restoration of cerebral blood flow can cause tissue injury, which is termed reperfusion injury. It is important to inhibit reperfusion injury to achieve greater brain protection. Because oxidative stress has been shown to activate mitogen-activated protein kinases (MAPKs), and because oxidative stress contributes to reperfusion injury, MAPK may be a potential target to inhibit reperfusion injury after brain ischemia. Here, we demonstrate that reperfusion after forebrain ischemia dramatically increases phosphorylation level of extracellular signal-regulated kinase 2 (ERK2) in the gerbil hippocampus. In addition, i.v. administration of U0126 (100-200 mg/kg), a specific inhibitor of MEK (MAPK/ERK kinase), protects the hippocampus against forebrain ischemia. Moreover, treatment with U0126 at 3 h after ischemia significantly reduces infarct volume after transient (3 h) focal cerebral ischemia in mice. This protection is accompanied by reduced phosphorylation level of ERK2, substrates for MEK, in the damaged brain areas. Furthermore, U0126 protects mouse primary cultured cortical neurons against oxygen deprivation for 9 h as well as nitric oxide toxicity. These results provide further evidence for the role of MEK/ERK activation in brain injury resulting from ischemia/reperfusion, and indicate that MEK inhibition may increase the resistance of tissue to ischemic injury.
Animals↗
Mitogen-activated protein kinase phosphorylates and targets inducible cAMP early repressor to ubiquitin-mediated destruction.
Inducible cAMP early repressor (ICER) is an important mediator of cAMP antiproliferative activity that acts as a putative tumor suppressor gene product. In this study, we examined the regulation of ICER protein by phosphorylation and ubiquitination in human choriocarcinoma JEG-3 and mouse pituitary AtT20 cells. We found that cAMP stabilized ICER protein by inhibiting the mitogen-activated protein kinase (MAPK) cascade. Activation of the MAPK pathway increased ICER phosphorylation. ICER phosphorylation was abrogated by inhibition of the MAPK pathway either by cAMP or directly by the MAPK inhibitor PD098059. The MAPKs extracellular signal-regulated kinases 1 and 2 physically interact with ICER and mediated the phosphorylation of ICER on a critical serine residue (Ser-41). A mutant form of ICER in which Ser-41 was substituted by alanine had a half-life 4-5 h longer than its wild-type counterpart. This alteration in stability was due to the inability of the Ser-41-mutant ICER to be efficiently ubiquitinated and degraded via the ubiquitin-proteasome pathway. These results present a novel cell signaling cross-talk mechanism at the cell nucleus between the MAPK and cAMP pathways, whereby MAPK targets a repressor of the cAMP-dependent gene expression for ubiquitination and proteasomal degradation.
Animals↗
Decreased mitogen activated protein kinase activities in congenital diaphragmatic hernia-associated pulmonary hypoplasia.
BACKGROUND/PURPOSE: The mechanisms that cause pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) currently are unknown. The authors proposed that the reduced size and immaturity of these lungs may be associated with differences in the levels of mitogen activated protein (MAP) kinase phosphorylation (extracellular signal regulated protein kinases, ERK-1 and -2). METHODS: ERK-1 activities were measured using immune-complex kinase assays on fetal whole-lung lysates obtained from both nitrofen and olive oil-treated (control) pregnant rats. In addition, ERK-1 and ERK-2 functional activities were estimated by semiquantitative Western blot analysis, using an antibody specific for the diphosphorylated (dp-ERK, activated) forms of the enzymes. RESULTS: ERK-1 activities, measured using immune-complex kinase assays, were reduced in CDH lungs compared with olive oil-treated controls (P <.02). In addition, dp-ERK-1 and dp-ERK-2 levels were found to be reduced in CDH lungs compared with controls (dp-ERK-1, P =.003; dp-ERK-2, P =.04), whereas ERK-1 and ERK-2 protein levels were unchanged. CONCLUSIONS: The lower values of ERK-1 activity and reduced amounts of dp-ERK-1 and dp-ERK-2 in lung tissue from CDH animals, suggests that ERK-1 and ERK-2 activities are reduced in pulmonary hypoplasia associated with CDH. The observed reduction in ERK-1 and ERK-2 activities implicates attenuated cell signaling upstream of the ERK-1 and -2 enzymes.
Animals↗
Sexual behaviour of heterosexual individuals with HIV infection naive for antiretroviral therapy in Italy.
BACKGROUND: Specific information about determinants of sexual behaviour of HIV infected heterosexuals, like injecting drug use (IDU), are essential to design interventions aimed at promoting safer sex practices. METHODS: We analysed data on sexual behaviour collected, between March 1997 and March 1999, through a self administered questionnaire among 1050 IDUs and 642 non-IDU heterosexuals enrolled in a prospective multicentre cohort study on the natural history of HIV infection. RESULTS: Among non-IDU heterosexuals, more women (48.5%) than men (25.1%) (p<0.001) reported that they were infected by HIV positive regular partners whose HIV status they were not aware of. Among the 1119 heterosexual males, one fifth reported having had more than 25 sexual partners during their lifetime. Condom use in the last sexual intercourse was more common among heterosexual IDUs (64.9%) than among non-IDU heterosexual males (58.3%) (p=0.05). Heterosexual IDU males were more likely (66.7%) than non-IDU heterosexuals (50.6%) to have an HIV negative partner (p<0.001). Of the 573 heterosexual females studied, 10.2% reported having had more than 25 lifetime sex partners. This proportion was higher among heterosexual IDUs (18.8%) than among non-IDU heterosexuals (4.3%) (p<0.001). Nearly 50% of the women in both groups reported having used a condom in the last intercourse. Almost 57% of heterosexual IDUs had a current HIV negative partner, compared with 34.9% non-IDU heterosexuals (p<0.001). In both sexes, the findings from univariate analysis were confirmed by multiple logistic regression analysis. CONCLUSIONS: This study identified some important differences, in both males and females, in sexual lifestyles according to injecting drug use (for example, in terms of HIV negative partners). This observation indicates the need to tailor HIV prevention messages according to history of injecting drug use.
Adult↗
Glycogen synthase kinase-3beta is a negative regulator of cardiomyocyte hypertrophy.
Hypertrophy is a basic cellular response to a variety of stressors and growth factors, and has been best characterized in myocytes. Pathologic hypertrophy of cardiac myocytes leads to heart failure, a major cause of death and disability in the developed world. Several cytosolic signaling pathways have been identified that transduce prohypertrophic signals, but to date, little work has focused on signaling pathways that might negatively regulate hypertrophy. Herein, we report that glycogen synthase kinase-3beta (GSK-3beta), a protein kinase previously implicated in processes as diverse as development and tumorigenesis, is inactivated by hypertrophic stimuli via a phosphoinositide 3-kinase-dependent protein kinase that phosphorylates GSK-3beta on ser 9. Using adenovirus-mediated gene transfer of GSK-3beta containing a ser 9 to alanine mutation, which prevents inactivation by hypertrophic stimuli, we demonstrate that inactivation of GSK-3beta is required for cardiomyocytes to undergo hypertrophy. Furthermore, our data suggest that GSK-3beta regulates the hypertrophic response, at least in part, by modulating the nuclear/cytoplasmic partitioning of a member of the nuclear factor of activated T cells family of transcription factors. The identification of GSK-3beta as a transducer of antihypertrophic signals suggests that novel therapeutic strategies to treat hypertrophic diseases of the heart could be designed that target components of the GSK-3 pathway.
Animals↗
Lipid oxidation deletes the nanodomain organization of artificial membranes.
Nanoscopic domains with different crystal structures have been induced in closed artificial membranes and have been directly imaged by atomic force microscopy at a spatial resolution better than 0.3 nm. These observations provide experimental evidence to the hydrophobic mismatching theory of lateral phase separation phenomena. Under oxidant conditions, the lipid-lipid assembly reorganizes into a new steady-state structure with disappearance of specific nanodomains. This finding may contribute to understanding the mechanism of peroxidative damage to membrane properties. In fact, alterations of specific modes of molecular conformation and packing may lead to perturbation of specific properties.
Lipids↗
Transfection of constitutively active mitogen-activated protein/extracellular signal-regulated kinase kinase confers tumorigenic and metastatic potentials to NIH3T3 cells.
Cellular growth and differentiation are controlled by multiple extracellular signals, many of which activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases. Components of the MAP kinase pathways also cause oncogenic transformation in their constitutively active forms. Moreover, expression of activated ras can confer metastatic potential upon some cells. Activation of MAP kinases requires phosphorylation of both Thr and Tyr in the catalytic domain by a family of dual-specificity kinases, called MEKs (MAP kinase/ERK kinase). MEK1 is activated by phosphorylation at Ser218 and Ser222 by Raf. Mutation of these two sites to acidic residues, specifically [Asp218], [Asp218, Asp222], and [Glu218, Glu222], results in constitutively active MEK1. Using these mutant variants of MEK1, we showed previously that transfection of NIH/3T3 or Swiss 3T3 cells causes morphological transformation and increases growth on soft agar, independent of ERK activity. The transformed cell lines show increased expression of matrix metalloproteinases 2 and 9 and cathepsin L, proteinases that have been implicated in the metastatic process. We tested NIH3T3 cells transfected with the [Asp218] or [Asp218, Asp222] for metastatic potential after i.v. injection into athymic mice. Parental 3T3 cells formed no tumors grossly or histologically. However, all MEK1 mutant transformants formed macroscopic metastases. Thus, like activated Ras, MEK1 can confer both tumorigenic and metastatic potential upon NIH3T3 cells. These results refine the mechanism through which ras could confer tumorigenic and metastatic potential (ie., the critical determinants of tumorigenic and metastatic potential are downstream of MEK1).
3T3 Cells↗
Serine-threonine protein kinase Akt does not mediate ischemic tolerance after global ischemia in the gerbil.
The protein kinase Akt/PKB has been implicated in antiapoptosis and neuronal survival. The authors now show that Akt is phosphorylated in the hippocampus during the early reperfusion period after 3.5 minutes bilateral carotid artery occlusion (BCAO) in the gerbil. Repeated sublethal ischemia induces ischemic tolerance, which is known as ischemic preconditioning. Ischemic preconditioning does not affect the amount of Akt protein, but rather decreases the phosphorylation of Akt at Ser-473 after 10 minutes reperfusion after 3.5 minutes BCAO. These results suggest that although Akt may play a role in neuronal survival after ischemia, it may not play a role in ischemic tolerance by preconditioning.
Animals↗
Different patterns of collagen-proteoglycan interaction: a scanning electron microscopy and atomic force microscopy study.
The extracellular matrix of unfixed, unstained rat corneal stroma, visualized with high-resolution scanning electron microscopy and atomic force microscopy after minimal preliminary treatment, appears composed of straight, parallel, uniform collagen fibrils regularly spaced by a three-dimensional, irregular network of thin, delicate proteoglycan filaments. Rat tail tendon, observed under identical conditions, appears instead made of heterogeneous, closely packed fibrils interwoven with orthogonal proteoglycan filaments. Pre-treatment with cupromeronic blue just thickens the filaments without affecting their spatial layout. Digestion with chondroitinase ABC rids the tendon matrix of all its interconnecting filaments while the corneal stroma architecture remains virtually unaffected, its fibrils always being separated by an evident interfibrillar spacing which is never observed in tendon. Our observations indicate that matrix proteoglycans are responsible for both the highly regular interfibrillar spacing which is distinctive of corneal stroma, and the strong interfibrillar binding observed in tendon. These opposite interaction patterns appear to be distinctive of different proteoglycan species. The molecular details of proteoglycan interactions are still incompletely understood and are the subject of ongoing research.
Animals↗
MEK1 protein kinase inhibition protects against damage resulting from focal cerebral ischemia.
The MEK1 (MAP kinase/ERK kinase)/ERK (extracellular-signal-responsive kinase) pathway has been implicated in cell growth and differentiation [Seger, R. & Krebs, E. G. (1995) FASEB J. 9, 726-735]. Here we show that the MEK/ERK pathway is activated during focal cerebral ischemia and may play a role in inducing damage. Treatment of mice 30 min before ischemia with the MEK1-specific inhibitor PD98059 [Alessi, D. R., Cuenda, A., Cohen, P. , Dudley, D. T. & Saltiel, A. R. (1995) J. Biol. Chem. 270, 27489-27494] reduces focal infarct volume at 22 hr after ischemia by 55% after transient occlusion of the middle cerebral artery. This is accompanied by a reduction in phospho-ERK1/2 immunohistochemical staining. MEK1 inhibition also results in reduced brain damage 72 hr after ischemia, with focal infarct volume reduced by 36%. This study indicates that the MEK1/ERK pathway contributes to brain injury during focal cerebral ischemia and that PD98059, a MEK1-specific antagonist, is a potent neuroprotective agent.
Animals↗
MEK1 activation rescues Jurkat T cells from Fas-induced apoptosis.
Although the protease cascade initiated by Fas (CD95, Apo-1) is well characterized, there remains little known about how kinase pathways may impact on Fas-mediated apoptosis. We recently observed that in T lymphocytes Fas strongly induced activation of JNK (c-Jun N-terminal kinase) but not of second messengers leading to activation of ERK (extracellular regulated kinase). Additionally, Fas-mediated apoptosis was significantly inhibited with PMA, a potent activator of the ERK signaling pathway. This suggested a model whereby activation of the ERK pathway might attenuate Fas-mediated apoptosis. This was confirmed in the current study by showing that activation of MEK1, the upstream regulator of ERK, reduces Fas-mediated apoptosis, whereas inhibition of MEK1 augments apoptosis by Fas. Furthermore, Fas-mediated apoptosis of Jurkat T cells is not affected by constitutively active or dominant negative variants that modulate the JNK pathway. These results demonstrate that Fas-induced JNK activation is not required for apoptosis by Jurkat T cells, but rather is more likely secondary to cell stress during the early phases of apoptosis. This is supported by the ability of the caspase blocker zVAD to inhibit both apoptosis and JNK activation by Fas.
Annexin A5↗
[Use of the TUT (Tension-free Vaginal Tape) in the treatment of female urinary stress incontinence. Preliminary results].
BACKGROUND: The Tension-free Vaginal Tape (TVT) represents the most recent technique for the treatment of genuine stress urinary incontinence (GSUI). The various number of surgical procedures proposed for the treatment of GSI very often do not lead to a complete remission of this pathology. The data from the literature show how TVT is a effective procedure for the treatment of female urinary incontinence. METHODS: Twenty-nine women with diagnosis of urinary incontinence underwent application of polypropilene band (TVT: tension-free vaginal tape) underneath the uretra, in order to treat this disorder. The procedure has been carried out in peripheral anesthesia. RESULTS: A complete remission of the urinary incontinence was obtained in 24 patients. In the remaining cases there was an improvement of the symptoms in two patients, whereas in two patients remained a secondary detrusor instability. In one case the external iliac vein was perforated thus requiring a surgical repair. CONCLUSION: The short surgical time, the feasibility of the procedure and the following short hospitalization made this technique well accepted either by the surgeons ang the patients. Moreover the possibility to carry out the procedure in peripheral anesthesia allows to have the collaboration of the patient. However this technique is not free of risks, how the serious complication we had can demonstrate.
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Inhibition of the p44/42 MAP kinase pathway protects hippocampal neurons in a cell-culture model of seizure activity.
Excessive release of glutamate and the subsequent influx of calcium are associated with a number of neurological insults that result in neuronal death. The calcium-activated intracellular signaling pathways responsible for this excitotoxic injury are largely unknown. Here, we report that PD098059, a selective inhibitor of the calcium-activated p44/42 mitogen-activated protein kinase (MAP kinase) pathway, reduces neuronal death in a cell-culture model of seizure activity. Dissociated hippocampal neurons grown chronically in the presence of kynurenate, a broad spectrum glutamate-receptor antagonist, and elevated amounts of magnesium exhibit intense seizure-like activity after the removal of these blockers of excitatory synaptic transmission. A 30-min removal of the blockers produced extensive neuronal death within 24 h as assayed by the uptake of trypan blue and the release of lactate dehydrogenase. Phospho-p44/42 MAP kinase immunoreactivity after 30 min of seizure-like activity was present in many neuronal somata and dendrites as well as some synaptic terminals, consistent with both the presynaptic and postsynaptic effects of this pathway. The addition of PD098059 (40 microM; EC50 = 10 microM) during a 30-min washout of synaptic blockers inhibited the phosphorylation of p44/42 MAP kinase and reduced both the trypan-blue staining (n = 13) and the release of lactate dehydrogenase (n = 16) by 73% +/- 18% and 75% +/- 19% (mean +/- SD), respectively. The observed neuroprotection could be caused by an effect of PD098059 on seizure-like events or on downstream signaling pathways activated by the seizure-like events. Either possibility suggests a heretofore unknown function for the p44/42 MAP kinase pathway in neurons.
Animals↗
Influence of indinavir and ritonavir on warfarin anticoagulant activity.
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Anti-HIV Agents↗
Study of elastic fiber organization by scanning force microscopy.
Elastic fibers of beef ligamentum nuchae were observed by atomic force microscopy and data compared with those obtained by conventional and freeze-fracture electron microscopy. Fresh isolated elastin fibers as well as thin sections of ligament fragments, which were fixed and embedded either in relaxed or in stretched conditions, were analysed. The results confirm that, at least in beef ligamentum nuchae, elastic fibers consist of beaded filaments which can be oriented by stretching in the direction of the force applied. Moreover, atomic force microscopy revealed that these beaded filaments are laterally connected by periodical bridges which become more pronounced upon stretching. The data clearly show that elastin molecules are organized in a rather ordered array, at least at the super-molecular level, and a depiction of the elastin organization in beef ligamentum nuchae is attempted.
Animals↗
Recognition of antigenic clusters of Candida albicans by T lymphocytes from human immunodeficiency virus-infected persons.
The fine specificity of the cellular immune response to Candida albicans (i.e., recognition of different antigenic components) between normal controls and human immunodeficiency virus-infected patients in various stages of disease was compared. C. albicans-specific T cells, enriched by antigen stimulation and interleukin-2 expansion, were challenged with antigenic fractions of different molecular weight obtained by SDS-gel fractionation of C. albicans extracts in the presence of autologous mononuclear cells as antigen-presenting cells. Proliferative responses showed similar patterns of reactivity between controls and category A and B seropositive subjects. Category C patients with concurrent C. albicans infections did not give rise to C. albicans-specific T cell lines, confirming the T cell defect. Patients without clinically evident C. albicans infection had a low but broad reactivity pattern of C. albicans-specific T cells. These results suggest that depletion of C. albicans-specific T cells, independent of their fine specificity, occurs along with disease progression.
Adult↗