Biomedical subjects

I Russo

Publications and source records attributed to I Russo.

At least 19 recordsLinked to original sources

PubMed2006

Insulin activates hypoxia-inducible factor-1alpha in human and rat vascular smooth muscle cells via phosphatidylinositol-3 kinase and mitogen-activated protein kinase pathways: impairment in insulin resistance owing to defects in insulin signalling.

AIMS/HYPOTHESIS: We previously demonstrated that insulin stimulates vascular endothelial growth factor (VEGF) synthesis and secretion via phosphatidylinositol-3 kinase (PI3-K) and mitogen-activated protein kinase (MAPK) pathways in vascular smooth muscle cells (VSMC) from humans and from insulin-sensitive lean Zucker fa/+ rats. We also showed that this effect is attenuated in VSMC from insulin-resistant obese Zucker fa/fa rats. As it is not known whether the effects of insulin on VEGF involve activation of hypoxia-inducible factor-1 (HIF-1), we aimed to evaluate: (1) whether insulin modulates HIF-1alpha protein synthesis and activity; (2) the insulin signalling pathways involved; and (3) the role of insulin resistance. METHODS: Using aortic VSMC taken from humans and Zucker rats and cultured in normoxia, the following were evaluated: (1) dose-dependent (0.5, 1, 2 nmol/l) and time-dependent (2, 4, 6 h) effects exerted by insulin on HIF-1alpha content in both nucleus and cytosol, measured by Western blots; (2) insulin effects on HIF-1 DNA-binding activity on the VEGF gene, measured by electrophoretic mobility shift assay; and (3) involvement of the insulin signalling molecules in these insulin actions, by using the following inhibitors: LY294002 (PI3-K), PD98059 (extracellular signal regulated kinase [ERK]), SP600125 (Jun N terminal kinase [JNK]), SB203580 (p38 mitogen-activated protein kinase) and rapamycin (mammalian target of rapamycin), and by detecting the insulin signalling molecules by Western blots. RESULTS: In aortic VSMC from humans and Zucker fa/+ rats cultured in normoxia insulin increases the HIF-1alpha content in cytosol and nucleus via dose- and time-dependent mechanisms, and HIF-1 DNA-binding activity on the VEGF gene. The insulin-induced increase of HIF-1alpha is blunted by the translation inhibitor cycloheximide, LY294002, PD98059, SP600125 and rapamycin, but not by SB203580. It is also reduced in Zucker fa/fa rats, which present an impaired ability of insulin to induce Akt, ERK-1/2 and JNK-1/2 phosphorylation. CONCLUSIONS/INTERPRETATION: These results provide a biological mechanism for the impaired collateral vessel formation in obesity.

Animals↗

PubMed2004

Impaired synthesis and action of antiaggregating cyclic nucleotides in platelets from obese subjects: possible role in platelet hyperactivation in obesity.

BACKGROUND: Subjects with central obesity exhibit platelet hyperactivity, which is involved in the atherosclerotic process and therefore can account for the increased risk of cardiovascular morbidity and mortality. The aim of the study was to evaluate whether alterations of platelet function in obesity involve synthesis and/or action of the two antiaggregating cyclic nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). MATERIALS AND METHODS: In platelets from 16 obese and 15 control subjects we investigated the influence on platelet responses to the Adenosine-5-diphosphate sodium salt (ADP) exerted by (i) prostacyclin analogue Iloprost (0.31-5 nmol L(-1)) and the cAMP analogue 8-bromo-cAMP (10-500 micro mol L(-1)); and by (ii) nitric oxide (NO) donor sodium nitroprusside (SNP) (5-100 micro mol L(-1)) and the cGMP analogue 8-bromo-cGMP (10-500 micro mol L(-1)). IC(50) (minimal concentration of each inhibitor necessary to reduce platelet response to ADP by half) was determined. Iloprost and SNP ability to increase cyclic nucleotides was also measured. RESULTS: Significantly greater IC(50) were observed in obese subjects than in healthy controls (1.59 +/- 0.16 vs. 0.80 +/- 0.08 nmol L(-1), P = 0.0001 for Iloprost, and 27.6 +/- 6.5 vs. 7.0 +/- 1.7 micro mol L(-1), P = 0.006, for SNP); when data from control and obese subjects were pooled together, IC(50) of Iloprost and SNP correlated with the homeostasis model assessment (HOMA IR), which is a parameter used to measure the insulin resistance (r = 0.588, P = 0.029 and r = 0.640, P = 0.006, respectively). Also the antiaggregating effect of 8-Br-cAMP and 8-Br-cGMP was smaller in the obese subjects. Finally, the ability of Iloprost to increase platelet cAMP and the ability of SNP to increase both cGMP and cAMP were reduced in obese subjects. CONCLUSIONS: Platelet resistance to the antiaggregating effects of prostacyclin and NO in obesity is attributable to impairment of cyclic nucleotide synthesis and action. As cyclic nucleotides are the main effectors of platelet antiaggregation, the resistance to them can account for platelet hyperactivity in obesity.

Adult↗

PubMed2004

Insulin activates vascular endothelial growth factor in vascular smooth muscle cells: influence of nitric oxide and of insulin resistance.

BACKGROUND: We aimed to evaluate whether insulin influences vascular endothelial growth factor (VEGF) synthesis and secretion in cultured vascular smooth muscle cells (VSMCs) via nitric oxide (NO) and whether these putative effects are lost in insulin-resistant states. MATERIALS AND METHODS: In VSMC derived from human arterioles and from aortas of insulin-sensitive Zucker fa/+rats and insulin-resistant Zucker fa/fa rats incubated with different concentrations of human regular insulin with or without inhibitors of phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3-K), mitogen-activated protein kinase (MAPK), nitric oxide synthase (NOS) and guanosine 3',5'cyclic monophosphate(cGMP)-dependent protein kinase (PKG), we measured protein expression (Western blot) and secretion (ELISA) of VEGF. RESULTS: We found that in VSMCs from humans and from insulin-sensitive Zucker fa/+rats, insulin increases VEGF protein expression and secretion, with mechanisms blunted by wortmannin and LY294002 (PI3-K inhibitors), PD98059 (MAPK inhibitor), L-NMMA (NOS inhibitor) and Rp-8pCT-cGMPs (PKG inhibitor). Also the NO donor sodium nitroprusside (SNP) and the cGMP analogue 8-Bromo-cGMP increase VEGF protein expression and secretion, with mechanisms inhibited by wortmannin and PD98059. The insulin effects on VEGF are impaired in VSMCs from Zucker fa/fa rats, which also present a reduced insulin ability to increase NO. CONCLUSIONS: In VSMCs from humans and insulin-sensitive Zucker fa/+rats: (i) insulin increases VEGF protein expression and secretion via both PI3-K and MAPK; (ii) the insulin effects on VEGF are mediated by nitric oxide. The insulin action on both nitric oxide and VEGF is impaired in VSMCs from Zucker fa/fa rats, an animal model of metabolic and vascular insulin-resistance.

Animals↗

PubMed2003

Insulin stimulates glucose transport via nitric oxide/cyclic GMP pathway in human vascular smooth muscle cells.

OBJECTIVE: In cultured human vascular smooth muscle cells, insulin increases cyclic GMP production by inducing nitric oxide (NO) synthesis. The aim of the present study was to determine whether in these cells the insulin-stimulated NO/cyclic GMP pathway plays a role in the regulation of glucose uptake. METHODS AND RESULTS: Glucose transport in human vascular smooth muscle cells was measured as uptake of 2-deoxy-d-[3H]glucose, cyclic GMP synthesis was checked by radioimmunoassay, and GLUT4 recruitment into the plasma membrane was determined by immunofluorescence. Insulin-stimulated glucose transport and GLUT4 recruitment were blocked by an inhibitor of NO synthesis and mimicked by NO-releasing drugs. Insulin- and NO-elicited glucose uptake were blocked by inhibitors of soluble guanylate cyclase and cyclic GMP-dependent protein kinase; furthermore, glucose transport was stimulated by an analog of cyclic GMP. CONCLUSIONS: Our results suggest that insulin-elicited glucose transport (and the corresponding GLUT4 recruitment into the plasma membrane) in human vascular smooth muscle cells is mediated by an increased synthesis of NO, which stimulates the production of cyclic GMP and the subsequent activation of a cyclic GMP-dependent protein kinase.

Biological Transport↗

PubMed2003

Natural history and clinical features of progressive supranuclear palsy: a clinical study.

Progressive supranuclear palsy (PSP or Steele-Richardson-Olszewski syndrome) is one of the most-common types of atypical parkinsonism. To characterize the natural history and the clinical features of PSP, we reviewed the records of 25 patients followed in our clinic since 1991, with a clinical diagnosis of PSP according to NINDS and Golbe criteria. Progressive onset of early bilateral bradykinesia and postural instability with falls during the 5th decade strongly support the diagnosis of PSP in our patients. Pseudobulbar symptoms are very common at onset and during the course of the illness.

Age of Onset↗

PubMed2003

In vitro effects of ultraviolet A on histamine release from human basophils.

BACKGROUND: As long-wave ultraviolet (UV) radiation penetrates the dermis, connective tissue cellular components and circulating blood cells can be possible targets for solar UVA. Basophils, involved in the effector phase of the inflammatory response, play a part in skin diseases such as chronic urticaria, psoriasis, atopic dermatitis, fixed drug eruption, allergic contact dermatitis, urticaria pigmentosa, systemic sclerosis and bullous pemphigoid. OBJECTIVE: The evaluation of the in vitro effect of UVA on histamine release from human basophils. METHODS: Basophils from healthy human volunteers were irradiated, respectively, with UVA at doses of 2.5, 5, 7.5, 10, 20 and 50 J/cm2 and then incubated with an anti-IgE serum. A fluorimetric technique was employed to determine histamine release from samples: (i) incubated with 2% HClO4 (complete lysis of basophils); (ii) irradiated with increasing doses of UVA; and (iii) unirradiated (controls). RESULTS: Histamine release was: 100% for HClO4 incubated basophils, 30% for unirradiated and anti-IgE incubated cells (controls) and 27%, 24%, 34%, 41%, 60% and 70% for basophils irradiated with UVA doses, respectively, of 2.5, 5, 7.5, 10, 20 and 50 J/cm2 and incubated with anti-IgE. Histamine releasability from irradiated samples was statistically significant (P < 0.05), in comparison with controls, at UVA doses equal to 5, 10, 20 and 50 J/cm2. CONCLUSIONS: UVA exerts, at least in vitro, a biphasic dose-dependent action on histamine release from human basophils incubated with an anti-IgE serum: at the lowest irradiation doses (< 5 J/cm2) it exerts an inhibitory effect and at the highest doses (> or = 10 J/cm2) histamine release increases significantly.

Basophils↗

PubMed2002

ALS-plus: 5 cases of concomitant amyotrophic lateral sclerosis and parkinsonism.

According to El Escorial criteria, amyotrophic lateral sclerosis (ALS), combined with other neurologic disorders, such as dementia and parkinsonism, is defined as ALS-plus. These overlaping syndromes are extremely rare. Here we report 5 cases (3 men, 2 women) of ALS-plus; mean age at the onset of symptoms was 67 years (range, 65-72). In 3 patients, motoneuronal signs preceded the onset of parkinsonian syndrome. In 4 cases, the clinical picture was characterized by the prevalence of motoneuronal signs. Parkinsonism was poorly responsive to L-dopa treatment in all patients. The clinical course did not differ from that expected in patients with only ALS. Our clinical observations and neuropathological reports of nigral neuronal loss in ALS patients suggest a common pathogenic mechanism underlying these disorders.

Aged↗

PubMed2002

[Detection of anomalous origin of left coronary artery by spiral CT as a cause of angina pectoris].

The anomalous origin of the left coronary artery can lead to angina pectoris, acute myocardial infarction or even sudden death, especially during exercise. We present a patient in whom the anomalous origin of the left coronary artery is from the right sinus of Valsalva, crossing between the aorta and the pulmonary trunk and causing ischemic chest pain. The anomaly was verified by a Spiral CT, as the coronary angiographic findings were not conclusive, particularly regarding the left course in relation to the major arteries. We suggest that Spiral CT is useful for detecting this kind of anomaly, particularly in clarifying the relationship between the left main coronary artery and the major arteries.

Adult↗

PubMed2001

Studies on inhibition of human platelet function by sodium nitroprusside. Kinetic evaluation of the effect on aggregation and cyclic nucleotide content.

In this study, we explored the ability of sodium nitroprusside to inhibit the aggregation of human platelets in platelet-rich plasma (PRP) and whole blood and its effects on intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP). The experiments investigated dose-dependent effects of nitroprusside starting from concentrations in the range of circulating levels achievable in vivo during drug administration in humans. Furthermore, we investigated the time-course of both antiaggregating action and the influence on cyclic nucleotide synthesis. Results showed that sodium nitroprusside inhibited the aggregation induced by adenosine 5-diphosphate (ADP) and collagen starting from concentration as low as 2 micromol/l. The IC(50) value for ADP-induced aggregation in PRP was 18.7+/-2.4 micromol/l. The inhibition of platelet aggregation showed a time-dependent behaviour and was not reversible within 90 min. The accumulation of intraplatelet cGMP in the presence of sodium nitroprusside exhibited a comparable time-course characterized by an early increase, a steady state and a late further increase. The time-course of cAMP synthesis was very similar to that of cGMP. Our data evidenced a long-lasting inhibition of platelet responses by sodium nitroprusside and excluded a desensitization of platelet guanylyl cyclase after 3-h exposure to nitric oxide (NO). Furthermore, they indicated a role of cAMP accumulation in the antiaggregating effects of nitroso donor: the simultaneous increase of intracellular content of cAMP and cGMP can synergize in the reduction of the platelet responses.

Adenosine Diphosphate↗

PubMed2001

17q21-qter trisomy is an indicator of poor prognosis in acute myelogenous leukemia.

A reciprocal translocation (9;11) is often found in acute myeloid leukemia (AML), mostly of the M5a type. We report a case of a child with AML, in whom t(9;11) was observed at diagnosis as the sole structural abnormality, together with trisomies 19 and 21. The diagnosis was AML evolving from a myelodysplastic syndrome (MDS), and the blast morphology was undifferentiated. Chemotherapy failed to induce morphological remission and the patient's condition soon worsened. A subclone appeared and expanded during the course of the disease, with an additional unbalanced translocation (1;17) leading to trisomy of the long arm of chromosome 17 (17q). The data available from the literature on acquired anomalies involving 17q and our observation led us to postulate a specific link between the gain of 17q and complete chemoresistance.

Acute Disease↗

PubMed2001

N-acetyl-L-cysteine exerts direct anti-aggregating effect on human platelets.

BACKGROUND: N-acetyl-L-cysteine, a thiol compound, has been shown to potentiate the inhibition of platelet aggregation exerted by organic nitrates and to increase the anti-aggregating effect of L-arginine, which promotes endogenous synthesis of nitric oxide (NO) acting as substrate of platelet constitutive nitric oxide synthase (NOS). It is not known whether this thiol can exert direct effects on platelet aggregability. MATERIALS AND METHODS: 14 healthy male volunteers provided platelet samples to investigate whether N-acetyl-L-cysteine directly influences platelet function and intraplatelet levels of 3',5' cyclic guanosine monophosphate (cGMP), which represents the second messenger involved in NO-induced antiaggregation. Some experiments were repeated in the presence of NOS inhibitor NG-monomethyl-L-arginine (L-NMMA), of nitric oxide-sensitive guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), of the selective cGMP phosphodiesterase inhibitor zaprinast and of calcium ionophores (A23187, ionomycin). RESULTS: N-acetyl-L-cysteine at 3000-6000 micromol L-1 decreases the responses of human platelets both in platelet-rich plasma (aggregation induced by adenosine 5-diphosphate) and in whole blood (aggregation induced by collagen). The anti-aggregating effect was prevented by preincubation with L-NMMA and guanylyl cyclase inhibitor ODQ. In resting platelets, N-acetyl-L-cysteine increased the levels of cGMP starting from a concentration of 3000 micromol L-1. Permeabilized platelets exhibited an increased sensitivity to the anti-aggregating effect of N-acetyl-L-cysteine. Also, cGMP phosphodiesterase inhibition or the increase in calcium availability, enhanced N-acetyl-L-cysteine effects on platelets. CONCLUSION: N-acetyl-L-cysteine exerts direct anti-aggregating effects through an increased bioavailability of platelet nitric oxide.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2000

Full cytogenetic characterization of a new neuroblastoma cell line with a complex 17q translocation.

Recent studies have shown that structural abnormalities of chromosome 17 resulting in gain of material are the most frequent genetic changes in neuroblastoma. We have established a new neuroblastoma cell line from a patient whose disease had evolved from stage 4s to 4, without evidence of deletion of the short arm of chromosome 1 and MYCN amplification, which are considered the most typical genetic indicators of aggressive disease. The cytogenetic study allowed a full characterization of the chromosome changes, and revealed a complex translocation of chromosome 17 leading to a derivative marker which may be described as follows: der(11)t(11;17)(p15;q12)t(11;17) (q22;q12). This resulted in a gain of part of the long arms of chromosome 17, which was recently associated with poor prognosis.

Animals↗

PubMed2000

Frequent estrogen and progesterone receptor immunoreactivity in renal angiomyolipomas from women with pulmonary lymphangioleiomyomatosis.

OBJECTIVE: To determine whether renal angiomyolipomas from women with pulmonary lymphangioleiomyomatosis (LAM) express estrogen receptor (ER) and progesterone receptor (PR). DESIGN: Retrospective study of archival tissue. PATIENTS: Twelve women with LAM and angiomyolipomas. SETTING: Fox Chase Cancer Center. INTERVENTIONS: ER and PR expression was studied using immunohistochemistry. The hormonal status of the patients at the time of resection of the angiomyolipoma was determined. RESULTS: Ten of the angiomyolipomas had ER immunoreactivity (83%), and all 12 had PR immunoreactivity (100%). The ER and PR positivity was in the smooth muscle component of the angiomyolipomas only. For five women, pulmonary LAM specimens were also available; two were ER positive (40%), and all five were PR positive (100%). All four angiomyolipomas from women receiving progesterone therapy were ER and PR positive. One tumor from a woman receiving tamoxifen was ER negative and strongly PR positive. One woman was pregnant; her tumor was ER and PR positive. CONCLUSIONS: ER and PR expression is frequent in renal angiomyolipoma cells from women with LAM. PR was more consistently present than ER in angiomyolipomas and in LAM. Our data suggest that angiomyolipoma growth could be affected by hormonal factors. If the growth of LAM-associated angiomyolipomas slows during hormonal therapy, there are two potential implications for LAM patients: first, angiomyolipoma size could serve as a measurable indication of response to hormonal therapy; and second, surgical removal of angiomyolipomas might be avoided in some cases.

Adult↗

PubMed2000

Whipple's disease. Description of a case and survey of the literature.

A case of Whipple's disease (WD) personally observed is described. A 28-year-old male was admitted to hospital for evaluation of weakness, intermittent fever and weight loss arisen since a month. On clinical investigation, he complained of vomit and diarrhea since three months. He had neither familial and personal past history of gastrointestinal diseases, nor any other important diseases. He denied use of drugs. Physical examination was negative. Laboratory findings showed anemia, low blood lymphocytes, low serum iron and total iron binding capacity, low total serum protein and low serum albumin and high level of ESR. Stool were negative for parasites and occult blood. Cultures of blood and urine were negative. Stool fat assay was > 7 g/24 h and D-xylose test showed a two-hour serum concentration < 25 mg/dl. Abdominal TC showed lumbo-aortic and mesenteric enlarged lymph nodes. An upper video endoscopy showed a duodenal lymphangectasia. Histological examination showed villar atrophy with massive infiltration of large PAS-positive diastase-resistant foamy cells. Ziehl-Nielsen staining was negative. WD was diagnosed and patient underwent therapy based on cotrimoxazole. This report emphasizes the difficulty to diagnose WD correctly, because of its rareness and clinical polymorphism. Recently, studies have identified a bacillus, Tropheryma whippelii, associated with WD, so that, in the next future, the diagnosis of WD will be faster and more accurate. Finally, it is important to administer antibiotics which can cross the blood brain barrier for at least one year, in order to prevent neurological relapse, often lethal.

Journal Article↗

PubMed1999

L-arginine modulates aggregation and intracellular cyclic 3,5-guanosine monophosphate levels in human platelets: direct effect and interplay with antioxidative thiol agent.

Platelet nitric oxide is involved in the control of aggregability via cyclic 3',5'-guanosine monophosphate synthesis. Since L-arginine provides a guanidino nitrogen group for nitric oxide synthesis through nitric oxide synthase activity, we tried to clarify whether an increased availability of this amino acid can directly modulate the response of human platelets. In our conditions, L-arginine (at 100-6000 micromol/L) was able to influence the response of human platelets stimulated with adenosine 5-diphosphate and collagen both in PRP and in whole blood. The anti-aggregating effect was not present when D-arginine was used. Permeabilized platelets exhibited an increased sensitivity to L-arginine. Also, an increased availability of Ca2+ enhanced L-arginine effect. L-arginine (at 120-500 micromol/L) increased cyclic 3',5'-guanosine monophosphate levels in resting platelets; the amino acid also determined an increase of cyclic 3',5'-guanosine monophosphate in platelets at the end of adenosine 5-diphosphate-induced aggregation. Nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine prevented L-arginine effects on aggregation and cyclic 3',5'-guanosine monophosphate synthesis. Phosphodiesterase III inhibitor milrinone and antioxidative thiol N-acetyl-L-cysteine enhanced the effect of L-arginine on cyclic 3',5'-guanosine monophosphate. In conclusion, L-arginine exerts inhibitory effects on human platelet response through a nitric oxide-dependent synthesis of cyclic 3',5'-guanosine monophosphate. A positive interplay on platelet response between L-arginine and milrinone or antioxidative thiol N-acetyl-L-cysteine was evidenced.

Acetylcysteine↗

PubMed1999

Prepubertal exposure to zearalenone or genistein reduces mammary tumorigenesis.

Prepubertal exposure to a pharmacological dose (500 mg kg(-1)) of the phyto-oestrogen genistein can reduce the incidence and multiplicity of carcinogen-induced mammary tumours in rats. However, such an exposure also disrupts the function of the hypothalamic-pituitary-gonadal axis, making it unsuitable for breast cancer prevention. We studied whether prepubertal exposure to genistein at a total body dose broadly comparable to the level typical of Oriental countries, approximately 1 mg kg(-1) body weight, affects mammary tumorigenesis. We also studied whether prepubertal exposure to zearalenone, a major source for phyto-oestrogens in the USA, influences breast cancer risk. Prepubertal rats were treated between postnatal days 7 and 20, with 20 microg (approximately 1 mg kg(-1) body weight) of either genistein or zearalenone. Zearalenone exposure significantly reduced both the incidence and multiplicity of mammary tumours induced by 7,12-dimethylbenz(a)anthracene (DMBA). Genistein exposure significantly reduced tumour multiplicity, but not tumour incidence, when compared with vehicle-treated animals. Furthermore, 60% of the tumours in the genistein group were not malignant, while all the tumours analysed for histopathology in the vehicle and zearalenone groups were adenocarcinomas. A higher number of differentiated alveolar buds, and lower number of terminal ducts, were present in the DMBA-treated mammary glands of the phyto-oestrogen exposed rats. The concentration of oestrogen receptor (ER) binding sites after the DMBA treatment was low in the mammary glands of all groups but a significantly higher proportion of the glands in the zearalenone exposed rats were ER-positive (i.e. ER levels > or = 5 fmol mg(-1) protein) than the glands of the vehicle controls. Our data suggest that a prepubertal exposure to a low dose of either zearalenone or genistein may protect the mammary gland from carcinogen-induced malignant transformation, possibly by increasing differentiation of the mammary epithelial tree.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1999

Incorporation of noncoded amino acids into the N-terminal domain 1-47 of hirudin yields a highly potent and selective thrombin inhibitor.

Hirudin is an anticoagulant polypeptide isolated from a medicinal leech that inhibits thrombin with extraordinary potency (Kd = 0.2-1.0 pM) and selectivity. Hirudin is composed of a compact N-terminal region (residues 1-47, cross-linked by three disulfide bridges) that binds to the active site of thrombin, and a flexible C-terminal tail (residues 48-64) that interacts with the exosite I of the enzyme. To minimize the sequence of hirudin able to bind thrombin and also to improve its therapeutic profile, several N-terminal fragments have been prepared as potential anticoagulants. However, the practical use of these fragments has been impaired by their relatively poor affinity for the enzyme, as given by the increased value of the dissociation constant (Kd) of the corresponding thrombin complexes (Kd = 30-400 nM). The aim of the present study is to obtain a derivative of the N-terminal domain 1-47 of hirudin displaying enhanced inhibitory potency for thrombin compared to the natural product. In this view, we have synthesized an analogue of fragment 1-47 of hirudin HM2 in which Val1 has been replaced by tert-butylglycine, Ser2 by Arg, and Tyr3 by beta-naphthylalanine, to give the BugArgNal analogue. The results of chemical and conformational characterization indicate that the synthetic peptide is able to fold efficiently with the correct disulfide topology (Cys6-Cys14, Cys16-Cys28, Cys22-Cys37), while retaining the conformational properties of the natural fragment. Thrombin inhibition data indicate that the effects of amino acid replacements are perfectly additive if compared to the singly substituted analogues (De Filippis V, Quarzago D, Vindigni A, Di Cera E, Fontana A, 1998, Biochemistry 37:13507-13515), yielding a molecule that inhibits the fast or slow form of thrombin by 2,670- and 6,818-fold more effectively than the natural fragment, and that binds exclusively at the active site of the enzyme with an affinity (Kd,fast = 15.4 pM, Kd,slow = 220 pM) comparable to that of full-length hirudin (Kd,fast = 0.2 pM, Kd,slow = 5.5 pM). Moreover, BugArgNal displays absolute selectivity for thrombin over the other physiologically important serine proteases trypsin, plasmin, factor Xa, and tissue plasminogen activator, up to the highest concentration of inhibitor tested (10 microM).

Amino Acid Sequence↗

PubMed1998

A telomere-independent senescence mechanism is the sole barrier to Syrian hamster cell immortalization.

Reactivation of telomerase and stabilization of telomeres occur simultaneously during human cell immortalization in vitro and the vast majority of human cancers possess high levels of telomerase activity. Telomerase repression in human somatic cells may therefore have evolved as a powerful resistance mechanism against immortalization, clonal evolution and malignant progression. The comparative ease with which rodent cells immortalize in vitro suggests that they have less stringent controls over replicative senescence than human cells. Here, we report that Syrian hamster dermal fibroblasts possess substantial levels of telomerase activity throughout their culture life-span, even after growth arrest in senescence. In our studies, telomerase was also detected in uncultured newborn hamster skin, in several adult tissues, and in cultured fibroblasts induced to enter the post-mitotic state irreversibly by serum withdrawal. Transfection of near-senescent dermal fibroblasts with a selectable plasmid vector expressing the SV40 T-antigen gene resulted in high-frequency single-step immortalization without the crisis typically observed during the immortalization of human cells. Collectively, these data provide an explanation for the increased susceptibility of rodent cells to immortalization (and malignant transformation) compared with their human equivalents, and provide evidence for a novel, growth factor-sensitive, mammalian senescence mechanism unrelated to telomere maintenance.

Animals↗