Biomedical subjects

L Sironi

Publications and source records attributed to L Sironi.

At least 19 recordsLinked to original sources

PubMed2006

Single nucleotide polymorphism discovery in the avian Tapasin gene.

Tapasin is a transmembrane glycoprotein located in the endoplasmic reticulum. Its function is to assist the assembly of major histocompatibility complex class I molecules. The chicken Tapasin gene includes 8 exons and is localized inside the major histocompatibility complex between the 2 class IIbeta genes. The aim of the current study was the estimation of single nucleotide polymorphism frequency within the avian Tapasin gene. The Tapasin gene sequence from exon 5 to exon 6 was amplified for the chicken, turkey, and pheasant, and sequences of different lengths were obtained. The sequence analysis based on PolyBayes identified 25 putative single nucleotide polymorphism sites when the 3 species were compared. The coding sequences were further translated and analyzed to identify amino acid substitutions. The results indicated that polymorphisms within this region of the gene was mainly observed in the heterozygous state. The level of conservation of the Tapasin gene sequence among species is likely to be related to the functional importance of the gene.

Amino Acid Sequence↗

PubMed2005

Myelin deterioration in Twitcher mice: motor evoked potentials and magnetic resonance imaging as in vivo monitoring tools.

We have used magnetic resonance imaging (MRI) and motor evoked potentials (MEPs) for monitoring disease progression within the CNS of the Twitcher mouse, the murine model for globoid cell leukodystrophy (GLD). GLD is a lysosomal storage disorder, resulting from galactocerebrosidase deficiency, causing central and peripheral myelin impairment, leading to death, usually during early infancy. Neuroradiological, electrophysiological, and pathological parameters of myelin maturation were evaluated in Twitcher mice between postnatal days 20 and 45. Healthy controls showed a gradual-appearance MRI T2-weighted hypointensity of the corpus callosum (CC) starting at about P30 and ending at about P37, whereas MRI of age-matched Twitcher mice showed a complete loss of the CC-related MRI signal. MEPs allowed the functional assessment of myelin maturation within corticospinal motor pathways and showed a progressive deterioration of MEPs in Twitcher mice with increased central conduction time (CCT; 5.12 +/- 0.49 msec at P27 to 6.45 +/- 1.96 msec at P32), whereas physiological CCT shortening was found in healthy controls (3.01 +/- 0.81 msec at P27 to 2.5 +/- 0.27 msec at P32). These findings were not paralleled by traditional histological stainings. Optical observation of Bielchowsky and Luxol fast blue-PAS stainings showed mild axonal/myelin deterioration of the Twitcher brain within this time frame. Our results demonstrate that serial MRI and MEP readings are sensitive evaluation tools for in vivo monitoring of dysmyelination in Twitcher mice and underscore their potential use for longitudinal evaluation of the therapeutic impact of gene and cell therapies on these animals.

Animals↗

PubMed2005

Neuroprotective effect of simvastatin in stroke: a comparison between adult and neonatal rat models of cerebral ischemia.

Statins, the most widely used lipid lowering drugs, have been demonstrated to play a protective role in stroke. Animal studies confirmed the observations obtained in clinical trials and provided additional data on the putative mechanism/s of action underlying this beneficial effect. We have shown that simvastatin reduced the size of the infarct to a different extend, according to the animal model used. Indeed, in the rat neonatal model of hypoxia/ischemia simvastatin affords protection only when is administered before the ischemic insult. In contrast, in adult rats bearing middle cerebral artery occlusion, simvastatin exerted its beneficial effect on brain injury when injected for 3 days either before or after induction of ischemia. Studies carried out to determine the therapeutic window of simvastatin demonstrated that the protective effect is observed after a single dose and when the drug is administered within 3-6 hours after ischemia. Simvastatin-dependent activation of eNOS has been claimed to be one of the main mechanisms responsible for neuroprotection. This hypothesis is confirmed in the adult animal model where eNOS is activated by either pre- or post- simvastatin treatment but is not supported by the data obtained in the neonate where eNOS activity is not affected by drug treatment. These observations suggest that the protective effect of simvastatin on stroke may be mediated by multiple mechanisms as can be expected by its pleiotropic effects.

Aging↗

PubMed2005

Multiexponential T2-relaxation analysis in cerebrally damaged rats in the absence and presence of a gadolinium contrast agent.

An analysis of the multiexponential relaxation of transverse nuclear magnetization with and without a gadolinium-based paramagnetic contrast agent in spontaneously hypertensive stroke-prone rats (SHR-SP) and in the rat model of ischemia induced by middle cerebral artery occlusion is described. From the multiexponential relaxation, the presence of two T(2) relaxation times in the range of 0.03-0.5 s, T(2A) (shortest) and T(2B) (longest), with very different relative weights (respectively, A and B), is evidenced. In our models of cerebral damage, the changes in A and B were more evident than those in T(2A) and T(2B). The two T(2) values were interpreted as belonging to water molecules in two different compartments; therefore, the difference between the damaged and normal regions revealed by means of standard T(2)-weighted images is suggested to be due to a different water distribution in the two compartments, rather than different T(2)'s. The T(2) relaxation in the SHR-SP stroke model is analyzed for the first time using a multiexponential method. The power of a detailed analysis of MRI relaxation times is confirmed by the correspondence between the revealed changes in T(2A), T(2B), A and B, and the known T(2)W and DWI results about blood-brain barrier functionality.

Animals↗

PubMed2001

Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores.

The kinetochore checkpoint pathway, involving the Mad1, Mad2, Mad3, Bub1, Bub3 and Mps1 proteins, prevents anaphase entry and mitotic exit by inhibiting the anaphase promoting complex activator Cdc20 in response to monopolar attachment of sister kinetochores to spindle fibres. We show here that Cdc20, which had previously been shown to interact physically with Mad2 and Mad3, associates also with Bub3 and association is up-regulated upon checkpoint activation. Moreover, co-fractionation experiments suggest that Mad2, Mad3 and Bub3 may be concomitantly present in protein complexes with Cdc20. Formation of the Bub3-Cdc20 complex requires all kinetochore checkpoint proteins but, surprisingly, not intact kinetochores. Conversely, point mutations altering the conserved WD40 motifs of Bub3, which might be involved in the formation of a beta-propeller fold devoted to protein-protein interactions, disrupt its association with Mad2, Mad3 and Cdc20, as well as proper checkpoint response. We suggest that Bub3 could serve as a platform for interactions between kinetochore checkpoint proteins, and its association with Mad2, Mad3 and Cdc20 might be instrumental for checkpoint activation.

Amino Acid Motifs↗

PubMed2001

Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint.

Mad2 is a key component of the spindle checkpoint, a device that controls the fidelity of chromosome segregation in mitosis. The ability of Mad2 to form oligomers in vitro has been correlated with its ability to block the cell cycle upon injection into Xenopus embryos. Here we show that Mad2 forms incompatible complexes with Mad1 and Cdc20, neither of which requires Mad2 oligomerization. A monomeric point mutant of Mad2 can sustain a cell cycle arrest of comparable strength to that of the wild-type protein. We show that the interaction of Mad2 with Mad1 is crucial for the localization of Mad2 to kinetochores, where Mad2 interacts with Cdc20. We propose a model that features the kinetochore as a 'folding factory' for the formation of a Mad2-Cdc20 complex endowed with inhibitory activity on the anaphase promoting complex.

Anaphase↗

PubMed2001

Proteins of rat serum, urine, and cerebrospinal fluid: VI. Further protein identifications and interstrain comparison.

We have investigated the biological fluids--serum, cerebrospinal fluid, and urine--of three strains of rats; the present data extend our database (also available on-line) and may be of interest for pharmacological and toxicological investigation. Specifically, we have defined reference maps of the major protein components in cerebrospinal fluid and urine. Compartment-specific isoforms were recognized for transferrin and transthyretin. Mass spectrometric data established the cleavage site of the signal peptide and identified the N-terminal blocking group of prostaglandin D synthase from rat cerebrospinal fluid. A previously undescribed member of the family of low molecular mass rat urinary proteins was characterized as containing a sequence similar, but not identical, to the N-terminal region of rat urinary protein-2 (RUP-2), and divergent from RUP-1.

Amino Acid Sequence↗

PubMed2001

Effects of androgen supplementation of hormone replacement therapy on the vascular reactivity of cerebral arteries.

OBJECTIVE: To determine the effect of the androgen supplementation of hormone replacement therapy (HRT) on the vascular reactivity of cerebral arteries. DESIGN: Open randomized study. SETTING: Healthy volunteers in an academic research environment. PATIENT(S): Forty postmenopausal women who were treated with sequential HRT (transdermal E2 50 microg/d + medroxyprogesterone acetate 10 mg/d for 12 days every other month) for > or =1 year and < or =5 years. INTERVENTION(S): Testosterone undecanoate (40 mg/d, p.o.) was randomly administered to 20 patients during ongoing HRT; the other 20 served as controls. Doppler evaluations of the internal carotid and middle cerebral arteries were performed together with lipid levels assessments. A visual analogue scale (VAS) was used to evaluate various parameters relating to sexual life and well-being. MAIN OUTCOME MEASURE(S): Pulsatility index (PI) of the arteries, VAS assessment of psychophysical well-being. RESULT(S): The administration of testosterone undecanoate during HRT induced an increase in the PI of the middle cerebral artery and a reduction of high-density lipoprotein cholesterol. Sexual desire and satisfaction were greatly improved. CONCLUSION(S): In postmenopausal women, androgen supplementation during HRT can partially counteract the beneficial effects of estrogens on cerebral vascular reactivity and lipid profiles, but sexual desire and satisfaction are greatly improved.

Administration, Cutaneous↗

PubMed2001

Effect of valsartan on angiotensin II-induced plasminogen activator inhibitor-1 biosynthesis in arterial smooth muscle cells.

Previous studies have shown that angiotensin II stimulates the synthesis of plasminogen activator inhibitor-1 in cultured vascular cells, which suggests that activation of the renin-angiotensin system may impair fibrinolysis. We have investigated the effects of angiotensin II and of valsartan, a recently developed angiotensin II antagonist that is highly specific and selective for the angiotensin II subtype 1 receptor, on plasminogen activator inhibitor-1 secretion by smooth muscle cells isolated from rat and human vessels. Angiotensin II induced a time- and concentration-dependent increase of plasminogen activator inhibitor activity in supernatants of rat aortic cells, which reached a plateau after 6 hours of incubation with 100 nmol/L angiotensin II (2.4+/-0.6-fold over control value; P:<0.001). The angiotensin II-induced plasminogen activator inhibitor activity was inhibited, in a concentration-dependent manner, by valsartan with an IC(50) value of 21 nmol/L. Valsartan fully prevented the angiotensin II-induced increase in plasminogen activator inhibitor-1 protein and mRNA. Furthermore, angiotensin II doubled the secretion of plasminogen activator inhibitor-1 by smooth muscle cells obtained from human umbilical and internal mammary arteries, and valsartan fully prevented it. Angiotensin II did not affect the secretion of tissue plasminogen activator antigen by any of the cell systems tested. Thus, valsartan effectively inhibits angiotensin II-induced plasminogen activator inhibitor-1 secretion without affecting that of tissue plasminogen activator in arterial rat and human smooth muscle cells.

Angiotensin II↗

PubMed2001

Acute-phase proteins before cerebral ischemia in stroke-prone rats: identification by proteomics.

BACKGROUND AND PURPOSE: A high degree of proteinuria has been reported in stroke-prone spontaneously hypertensive rats (SHRSP). We studied the effect of salt loading on the detailed protein pattern of serum and urine in 3 rat strains: Wistar-Kyoto, spontaneously hypertensive rats, and SHRSP, an inbred animal model for a complex form of cerebrovascular disorder resembling the human disease. METHODS: Rats were given a permissive diet and received 1% NaCl in drinking water. The protein pattern in body fluids was assessed over time by 2-dimensional electrophoretic analysis. Brain alterations were monitored by MRI and histology. RESULTS: Several proteins were excreted in urine after weeks of treatment and in advance of stroke: transferrin, hemopexin, albumin, alpha(2)-HS-glycoprotein, kallikrein-binding protein, alpha(1)-antitrypsin, Gc-globulin, and transthyretin. Markers of an inflammatory response, including very high levels of thiostatin, were detected in the serum of SHRSP at least 4 weeks before a stroke occurred. CONCLUSIONS: In SHRSP subjected to salt loading, an atypical inflammatory condition and widespread alterations of vascular permeability developed before the appearance of anomalous features in the brain detected by MRI. Urinary concentrations of each of the excreted serum proteins correlated positively with time before stroke occurred.

Acute-Phase Proteins↗

PubMed2001

Transcriptional regulation of plasminogen activator inhibitor type 1 gene by insulin: insights into the signaling pathway.

Impairment of the fibrinolytic system, caused primarily by increases in the plasma levels of plasminogen activator inhibitor (PAI) type 1, are frequently found in diabetes and the insulin-resistance syndrome. Among the factors responsible for the increases of PAI-1, insulin has recently attracted attention. In this study, we analyzed the effects of insulin on PAI-1 biosynthesis in HepG2 cells, paying particular attention to the signaling network evoked by this hormone. Experiments performed in CHO cells overexpressing the insulin receptor indicate that insulin increases PAI-1 gene transcription through interaction with its receptor. By using inhibitors of the different signaling pathways evoked by insulin-receptor binding, it has been shown that the biosynthesis of PAI-1 is due to phosphatidylinositol (PI) 3-kinase activation, followed by protein kinase C and ultimately by mitogen-activated protein (MAP) kinase activation and extracellular signal-regulated kinase 2 phosphorylation. We also showed that this pathway is Ras-independent. Transfection of HepG2 cells with several truncations of the PAI-1 promoter coupled to a CAT gene allowed us to recognize two major response elements located in the regions between -804 and -708 and between -211 and -54. Electrophoretic mobility shift assay identified three binding sites for insulin-induced factors, all colocalized with putative Sp1 binding sites. Using supershifting antibodies, the binding of Sp1 could only be confirmed at the binding site located just upstream from the transcription start site of the PAI-1 promoter. A construct comprising four tandem repeat copies of the -93/-62 region of the PAI-1 promoter linked to CAT was transcriptionally activated in HepG2 cells by insulin. These results outline the central role of MAP kinase activation in the regulation of PAI-1 induced by insulin.

Base Sequence↗

PubMed2001

Magnesium inhibits arterial thrombi after vascular injury in rat: in vivo impairment of coagulation.

Magnesium deficiency is associated with a high frequency of cardiac arrhythmia, hypertension and sudden ischemic death. We investigated the if vivo effects of intravenous magnesium administration in a rat model of chemically induced (FeCl3) carotid thrombosis. The infusion of magnesium sulfate (MgSO4) before the topical application of FeCl5 prevented thrombus formation at concentrations of 0.3 M and 0.6 M, and delayed it even at 0.15 M. Similar results were obtained with MgCl2. The infusion of MgSO4 0.6 M seven minutes after FeCl3 application delayed but did not prevent thrombus formation. MgSO4 slightly reduced platelet aggregation ex vivo without affecting plasma clotting tests, but in vivo blood clotting time was markedly prolonged (tail transection method), thus indicating profoundly impaired coagulation. These data provide a rationale for the use of magnesium as an antithrombotic agent. but its pharmacological effect critically depends on the timing of administration.

Animals↗

PubMed2000

Temporal trends of stroke in Valle d'Aosta, Italy. Incidence and 30-day fatality rates.

The objective of this study was to evaluate temporal changes of stroke in an Italian community by comparing the present incidence rates with those reported in the same area for 1989. The two studies were conducted by the same research group and met almost all the criteria proposed for an "ideal" stroke incidence study. The annual incidence rate per 1000 inhabitants increased (p < 0.01) by 29%, from 2.23 (95% CL, 1.96-2.50) in 1989 to 2.89 (95% CL, 2.58-3.20) in 1997. No statistically significant change was found when these rates were adjusted to the 1991 Italian population. The overall incidence rate was 2.40 (95% CL, 2.14-2.66) in 1989 and 2.65 (95% CL, 2.39-2.91) in 1997. The thirty-day case fatality rate declined dramatically (p < 0.001) from 31% (95% CL, 26-36) to 20% (95% CL, 16-24) between 1989 and 1997. Ageing of the population and better identification of cases could explain the high incidence rate, whereas the decrease of fatality rate may be due to a general improvement in acute care and inclusion of milder cases.

Adolescent↗

PubMed2000

Fluvastatin inhibits basal and stimulated plasminogen activator inhibitor 1, but induces tissue type plasminogen activator in cultured human endothelial cells.

The effects of fluvastatin, a synthetic hydroxymethylglutaryl coenzyme A (HMG-CoA) inhibitor, on the biosynthesis of tissue plasminogen activator (t-PA) and of its major physiological inhibitor (plasminogen activator inhibitor type 1, PAI-1) were investigated in cultured human umbilical vein endothelial cells (HUVEC). Fluvastatin (0.1 to 2.5 microM), concentration-dependently reduced the release of PAI-1 antigen by unstimulated HUVEC, subsequent to a reduction in PAI-1 steady-state mRNA levels and de novo protein synthesis. In contrast, it increased t-PA secretion. The drug also reduced PAI-1 antigen secreted in response to 10 microg/ml bacterial lipopolysaccharide (LPS), 100 U/ml tumour necrosis factor alpha (TNFalpha) or 0.1 microM phorbol myristate acetate (PMA). Mevalonate (100 microM), a precursor of isoprenoids, added to cells simultaneously with fluvastatin, suppressed the effect of the drug on PAI-1 both in unstimulated and stimulated cells as well as on t-PA antigen. Among intermediates of the isoprenoid pathway, all-transgeranylgeraniol (5 microM) but not farnesol (10 microM) prevented the effect of 2.5 microM fluvastatin on PAI-1 antigen, which suggests that the former intermediate of the isoprenoid synthesis is responsible for the observed effects.

Cells, Cultured↗

PubMed1999

Incidence of xenobiotics among drivers killed in single-vehicle crashes.

The authors have performed a study of single-vehicle crashes (SVCs) in order to verify a correlation between the loss of vehicle control and the presence of drugs in the body. Overall, 129 cases were recorded and occurred in the catchment area of the Institute of Legal Medicine in Milan between 1986 to 1996. Among the 129 cases under study, respectively 121 men and eight women, 101 were car-drivers and 28 motor-cyclists. The median age was equal to 29 years, while the average age to 32.0 years (range 15-65 years). Fifty eight cases (45.0%) were "positive" for the presence of ethanol > or = 0.8 g/l or other drugs. The sample of "positive cases" was studied according to sex, age, day, hour and type of vehicle. Considering the cases with presence of ethanol, although under the legal limit (20 cases), the total amount of cases (78) becomes even more consistent. The amount of ethanol was found to be respectively 0.34 g/l in daily drivers and 0.87 g/l in nightly drivers (p < 0.01). Our considerations confirm the importance of toxicological analyses in the forensic investigation of traffic deaths being the sample under study recorded following criteria which minimised other possible factors effecting road accidents.

Accidents, Traffic↗

PubMed1999

Blood flow in the internal carotid and middle cerebral arteries: effects of continuous oral conjugated equine estrogens administration with monthly progestogen supplementation on postmenopausal women.

OBJECTIVE: This study was designed in order to evaluate the effect of conjugated equine estrogens (CEE) on internal carotid and middle cerebral artery blood flow in postmenopausal women. DESIGN: Thirty-four healthy postmenopausal women with intact uteri were randomly divided into two groups of 17 subjects each. The first group was treated for 24 weeks with continuous CEE medication (0.625 mg daily) and cyclical supplementations of 5 mg/day of medrogestone acetate, given on the last 12 days of every 4-week period (Prempak, Wyeth, Italy). The second group received no treatment. The pulsatility indices (PI) of both the internal carotid artery and middle cerebral artery were measured. RESULTS: In the treated group, the PI of the interior carotid artery and MCA was reduced from respectively 0.736 (0.016) and 0.745 (0.009) at baseline, to 0.669 (0.021) and 0.670 (0.011) after 24 weeks (p = 0.01); in the control group, the PI values remained unchanged. The between-group difference for both arteries was significant (p < 0.01). CONCLUSIONS: The administration of CEE with cyclical medrogestone supplementation to postmenopausal women induces a statistically significant reduction in the PI of cerebral arteries.

Administration, Oral↗

PubMed1999

Oxidized low density lipoprotein suppresses expression of inducible cyclooxygenase in human macrophages.

Atherogenesis involves several aspects of chronic inflammation and wound healing. Indeed, the atheroma is considered a special case of tissue response to injury. Injurious stimuli may include lipoproteins trapped within lesions where protein and lipid moieties have undergone chemical modifications. We have studied the effect of oxidized low density lipoproteins (ox-LDL) on inducible cyclooxygenase (Cox-2) in human monocyte-derived macrophages exposed to bacterial lipopolysaccharide (LPS). Levels of both Cox-2 and constitutive cyclooxygenase (Cox-1) were assessed using Western blot analysis. Prior incubation of macrophages with ox-LDL resulted in a strong inhibition of Cox-2 induced by LPS, without effect on Cox-1. The inhibitory effect was dependent on ox-LDL concentration and its onset was early in time (already detectable 1 hour after macrophage exposure to ox-LDL). Native LDL, and other forms of modified LDL, were without effect. The inhibition was dependent on endocytosis of ox-LDL and could be reproduced using the lipid extract from ox-LDL. Lysophosphatidylcholine, 7beta-hydroxycholesterol, and 7-oxocholesterol failed to mimic the inhibition, but oxidized arachidonic acid-containing phospholipids, produced by autoxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, markedly inhibited Cox-2. The observation that ox-LDL downregulates Cox-2 in human macrophages may explain the fact that, within atheromata, the transformation of macrophages into foam cells results in attenuation of the inflammatory response, thus contributing to progression of atherogenesis.

Cells, Cultured↗

PubMed1998

The effect of tamoxifen and transdermal 17beta-estradiol on cerebral arterial vessels: a randomized controlled study.

OBJECTIVES: Our objective was to study the effects of tamoxifen on cerebral arterial reactivity. STUDY DESIGN: We studied the reactivity of both the internal carotid artery and the middle cerebral artery during a 12-month period of administration of either oral tamoxifen or transdermal estradiol or no treatment. A total of 45 healthy postmenopausal women who had undergone hysterectomy were followed up. Patients were randomly allocated to treatment with either oral tamoxifen 20 mg/day or transdermal estradiol 50 microg/day or nothing (15 patients in each group). They all underwent Doppler examinations of the internal carotid artery and middle cerebral artery at the beginning of the study and after 2, 6, and 12 months of treatment. The pulsatility index was measured. RESULTS: In the women given transdermal estradiol the pulsatility index of both the internal carotid artery and the middle cerebral artery was significantly reduced compared with that in the controls. Tamoxifen did not induce variations of pulsatility index in either artery during all the study period. The difference between the effect of the two drugs on the pulsatility index of both arteries was highly significant. CONCLUSIONS: Our findings demonstrate that tamoxifen does not cause any variation in the pulsatility index of cerebral arteries. The action of transdermal estradiol on the pulsatility index of cerebral arteries in postmenopausal women is the expression of a generalized action of estrogens on arterial vessels, and if this expression plays a role in the protective effect of hormone replacement therapy on risk of cardiovascular disease, tamoxifen treatment in healthy postmenopausal women should be considered with renewed caution.

Administration, Cutaneous↗