pp56Lck mediates TCR zeta-chain binding to the microfilament cytoskeleton

The TCR zeta-chain (zeta) on mature murine T lymphocytes binds to the microfilament cytoskeleton in response to Ag receptor ligation. Here, we report the role of Src family kinases in zeta-cytoskeletal binding, using mutant mice and a cell-free model system. Binding of zeta to actin in the cell-free system has a specific requirement for ATP and divalent cations, with an apparent Michaelis-Menton constant for ATP in the millimolar range, and can be disrupted by either EDTA or the microfilament poison, cytochalasin D, suggesting that microfilaments provide the structural framework for an active process involving cellular kinases. Indeed, tyrosine-phosphorylated zeta is a predominant form of the zeta-chain bound to polymerized actin, while challenge with alkaline phosphatase prevents zeta-chain association in solution and releases zeta-chain from the bound state. Phosphorylated Src-family kinase pp56Lck also associates with membrane skeleton upon TCR engagement and is a component of the reconstituted cytoskeletal pellet. Zeta-chain phosphorylation and zeta-cytoskeletal binding are abrogated in cell lysates with reduced levels of pp56Lck and in activated mutant murine T cells lacking pp56Lck, implicating pp56Lck as the kinase involved in zeta-chain tyrosine phosphorylation and zeta-cytoskeletal binding. Finally, recombinant Lck Src homology 2 domain preferentially inhibits reconstituted zeta-cytoskeleton association, suggesting that zeta-microfilament binding is dependent on interactions between phosphorylated tyrosine residues in zeta-chain activation motifs and the Src homology 2 domain of the Lck protein tyrosine kinase.     

Biochemical and pharmacological properties of SANORG 34006, a potent and long-acting synthetic pentasaccharide

SANORG 34006 is a new sulfated pentasaccharide obtained by chemical synthesis. It is an analog of the "synthetic pentasaccharide" (SR 90107/ ORG 31540) which represents the antithrombin (AT) binding site of heparin. SANORG 34006 showed a higher affinity to human AT than SR 90107/ORG 31540 (kd = 1.4 +/- 0.3 v 48 +/- 11 nmol/L), and it is a potent and selective catalyst of the inhibitory effect of AT on factor Xa (1,240 +/- 15 anti-factor Xa U/mg v 850 +/- 27 anti-factor Xa U/mg for SR 90107/ORG 31540). In vitro, SANORG 34006 inhibited thrombin generation occurring via both the extrinsic and intrinsic pathway. After intravenous (IV) or subcutaneous (SC) administration to rabbits, SANORG 34006 displayed a long-lasting anti-factor Xa activity and inhibition of thrombin generation (TG) ex vivo. SANORG 34006 was slowly eliminated after IV or SC administration to rats, rabbits, and baboons, showed exceptionally long half-lives (between 9.2 hours in rats and 61.9 hours in baboons), and revealed an SC bioavailability near 100%. SANORG 34006 displayed antithrombotic activity by virtue of its potentiation of the anti-factor Xa activity of AT. It strongly inhibited thrombus formation in experimental models of thromboplastin/stasis-induced venous thrombosis in rats (IV) and rabbits (SC) (ED50 values = 40.0 +/- 3.4 and 105.0 +/- 9.4 nmol/kg, respectively). The duration of its antithrombotic effects closely paralleled the ex vivo anti-factor Xa activity. SANORG 34006 enhanced rt-PA-induced thrombolysis and inhibited accretion of 125I-fibrinogen onto a preformed thrombus in the rabbit jugular vein suggesting that concomitant use of SANORG 34006 during rt-PA therapy might be helpful in facilitating thrombolysis and preventing fibrin accretion onto the thrombus under lysis. Contrary to standard heparin, SANORG 34006 did not enhance bleeding in a rabbit ear incision model at a dose that equals 10 times the antithrombotic ED50 in this species and, therefore, exhibited a favorable therapeutic index. We suggest that SANORG 34006 is a promising compound in the treatment and prevention of various thrombotic diseases.     

5-hydroxytryptamine1A receptor-mediated effects on adenylate cyclase and nitric oxide synthase activities in rat ventral prostate

AIM: To investigate the patterns of expression of transforming growth factor alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) in squamous metaplasia and squamous cell carcinomas of the urinary bladder with and without schistosomiasis. METHODS: Immunohistochemical study of the expression of TGF-alpha and EGFR in squamous metaplasias (n = 12) and various grades of squamous cell carcinomas (n = 21) of the bladder with and without schistosomiasis. RESULTS: Focal cytoplasmic and membranous positivity for EGFR and TGF-alpha was seen in all cases of squamous metaplasia. The markers were diffusely coexpressed in a concordant pattern in areas of hyperplastic keratinising squamous metaplasia. A similar pattern of positivity was seen in verrucous carcinomas (n = 2) and well differentiated squamous carcinomas (n = 6). Progressive loss of differentiation was associated with increasing loss of EGFR staining while TGF-alpha staining was retained. Squamous cell carcinoma in situ (n = 2) showed focal positivity for TGF-alpha and EGFR. There were no differences in staining patterns between cases with and without schistosomiasis. CONCLUSIONS: The coexpression of TGF-alpha and EGFR by well differentiated squamous cell carcinomas and hyperplastic keratinising squamous metaplasia is consistent with the active regulatory role exerted by this autocrine loop. There is regional absence of expression of EGFR but not of TGF-alpha in squamous cell carcinomas of lesser differentiation, suggesting heterogeneity of such control in these tumours. The focal expression of the two markers in squamous cell carcinomas in situ indicates a possible second pathway of oncogenesis for less differentiated tumours. These observations may have important implications for the effectiveness of putative growth factor based treatments.     

Cytotoxicity of curcuminoids and some novel compounds from Curcuma zedoaria

Bioassay-directed fractionation of an EtOH extract of Curcuma zedoaria led to isolation of an active curcuminoid, which was identified as demethoxycurcumin (2) by comparison of its 1H and 13C NMR spectra with literature data and by direct comparison with synthetic material. Curcumin (1) and bisdemethoxycurcumin (3) were also obtained. Curcuminoids (1-3) were synthesized and demonstrated to be cytotoxic against human ovarian cancer OVCAR-3 cells. The observed CD50 values of 1, 2, and 3 were 4.4, 3.8, and 3.1 microg/mL, respectively. Three additional novel compounds, 3, 7-dimethylindan-5-carboxylic acid (4), curcolonol (5), and guaidiol (6), were also isolated from the EtOH extract. The structures and relative stereochemistry of 4-6 were determined by spectroscopic methods and X-ray crystallographic analysis.     

Persistent hyperinsulinemic hypoglycemia in the newborn and infants

Persistent hyperinsulinemic hypoglycaemia of infancy (PHHI) is the most frequent cause of hypoglycaemia in infancy. Clinical presentation is heterogeneous, with variable onset of hypoglycaemia and response to diazoxide, and presence of sporadic or familial forms. Underlying histopathological lesions can be focal or diffuse. Focal lesions are characterised by focal hyperplasia of pancreatic islet-like cells, whereas diffuse lesions implicate the whole pancreas. The distinction between the two forms is important because surgical treatment and genetic counselling are radically different. Focal lesions correspond to somatic defects which are totally cured by limited pancreatic resection, whereas diffuse lesions require a subtotal pancreatectomy exposing to high risk of diabetes mellitus. Diffuse lesions are due to functional abnormalities involving several genes and different transmission forms. Recessively inherited PHHI have been attributed to homozygote mutations for the beta-cell sulfonylurea receptor (SUR1) or the inward-rectifying potassium-channel (Kir6.2) genes. Dominantly inherited PHHI can implicate the glucokinase gene, particularly when PHHI is associated with diabetes, the glutamate dehydrogenase gene when hyperammonaemia is associated, or another locus.     

Chromosomal instability is correlated with telomere erosion and inactivation of G2 checkpoint function in human fibroblasts expressing human papillomavirus type 16 E6 oncoprotein

Cell cycle checkpoints and tumor suppressor gene functions appear to be required for the maintenance of a stable genome in proliferating cells. In this study chromosomal destabilization was monitored in relation to telomere structure, lifespan control and G2 checkpoint function. Replicative senescence was inactivated in secondary cultures of human skin fibroblasts by expressing the human papillomavirus type 16 (HPV-16) E6 oncoprotein to inactivate p53. Chromosome aberrations were enumerated during in vitro aging of isogenic control (F5neo) and HPV-16E6-expressing (F5E6) fibroblasts. We found that structural and numerical aberrations in chromosomes were significantly increased in F5E6 cells during aging in vitro and fluorescence in situ hybridization (FISH) analysis using chromosome-specific probes demonstrated the occurrence of rearrangements involving chromosome 4 and 6 in genetically unstable F5E6 cells. Flow cytometry and karyotypic analyses revealed increased polyploidy and aneuploidy in F5E6 cells only at passages > 16, although these cells displayed defective mitotic spindle checkpoint function associated with inactivation of p53 at passages 5 and 16. G2 checkpoint function was confirmed to be gradually but progressively inactivated during in vitro aging of E6-expressing cells. Aging of F5neo fibroblasts was documented during in vitro passaging by induction of a senescence-associated marker, pH 6.0 lysosomal beta-galactosidase. F5E6 cells displayed extension of in vitro lifespan and did not induce beta-galactosidase at high passage. Erosion of telomeres during in vitro aging of telomerase-negative F5neo cells was demonstrated by Southern hybridization and by quantitative FISH analysis on an individual cell level. Telomeric signals diminished continuously as F5neo cells aged in vitro being reduced by 80% near the time of replicative senescence. Telomeric signals detected by FISH also decreased continuously during aging of telomerase-negative F5E6 cells, but telomeres appeared to be stabilized at passage 34 when telomerase was expressed. Chromosomal instability in E6-expressing cells was correlated (P < 0.05) with both loss of telomeric signals and inactivation of G2 checkpoint function. The results suggest that chromosomal stability depends upon a complex interaction among the systems of telomere length maintenance and cell cycle checkpoints.     

T cell apoptosis in human heart allografts: association with lack of co-stimulation?

It is unclear whether the intracardial immune reactivity after heart transplantation influences the peripheral immunological status (activation or nonresponsiveness) of the patient. Co-stimulation and activation-induced cell death (AICD) or apoptosis play an important role in determining the balance between lymphocyte reactivity and nonreactivity. Therefore, we studied the expression of co-stimulatory molecules and the process of apoptosis in biopsies of human heart allografts, using immunohistochemistry. Although a normal expression of co-stimulatory molecules on antigen-presenting cells was observed, the expression of their counter-structures on T cells was absent. This may be due to chronic T cell activation, which can lead to the induction of apoptosis via the Fas/Fas ligand pathway. In the infiltrates, a considerable percentage of the lymphocytes, but not the macrophages, were apoptotic. Apoptosis was confirmed by DNA fragmentation analysis. Increased numbers of Bax-expressing versus decreased numbers of Bcl2-expressing lymphocytes in comparison with normal lymphoid tissue confirmed a imbalance in favor of apoptosis. Apoptosis was biased towards CD4+ T cells (65.7% versus 26.6% in CD8+ T cells). Fas was expressed on most of the infiltrating cells. Fas ligand expression was also observed, not only on most of the T cells but also on all macrophages. Because macrophages were often detected in close contact with T cells, they may play a role in T cell regulation via the Fas/Fas ligand pathway. This study indicates that, during rejection, not only is tissue damage induced by infiltrating T cells, but also the infiltrating lymphocytes themselves are actively down-regulated (eg, AICD) by one another and by macrophages in the infiltrate. This regulatory process may affect the immunological status of the patient after heart transplantation.     

Symptoms of depression among community-dwelling elderly African-American and white older adults

BACKGROUND: Few studies have explored the variance in individual symptoms by race in older adults. METHODS: Data were analysed from the Duke site of the Established Populations for Epidemiologic Studies of the Elderly (EPESE), a community sample of persons 65 years-of-age and older, 54% of whom were African-Americans. Of the 3401 subjects with adequate data on depressive symptomatology, confirmatory factor analysis and LISREL were first used to confirm the presence of the factor structure previously reported for the CES-D. Next, bivariate analysis was performed to determine the prevalence of individual symptoms by race. Finally, LISREL analysis was performed to control for potential confounding variables. RESULTS: When bivariate comparisons of specific symptoms by race were explored, African-Americans were more likely to report less hope about the future, poor appetite, difficulty concentrating, requiring more effort for usual activities, less talking, feeling people were unfriendly, feeling disliked by others and being more 'bothered' than usual. When LISREL analyses were applied to these data (controlling for education, income, cognitive impairment, chronic health problems and disability and other factors) racial differences in somatic complaints and life satisfaction disappeared, yet differences in interpersonal relations persisted. CONCLUSIONS: This study confirms earlier findings of minimal overall differences in symptom frequency between African-American and non-African-American community-dwelling older adults in controlled studies.     

Bibliometric index

The Institute of Scientific information has edited an index to evaluate the diffusion of scientific articles. It is based on the fact that the more an article is cited as a reference in other articles, the more it is considered to be important, and the higher the coefficient attributed to the journal in which it was originally published. The impact factor takes into account the average number of times which a journal is mentioned for recent articles published in a given year. This index is an attempt to quantify the notoriety of scientific journal for all scientific medical specialties.     

Surface structure of human mucin using X-ray photoelectron spectroscopy

The stimulating effect of antiparkinsonian drugs, talipexole and bromocriptine, on the striatal postsynaptic dopamine receptors were studied by measuring contralateral rotational behavior in rats. The nigro-striatal dopamine system of rats was degenerated by unilateral injection of 6-hydroxydopamine (6-OHDA, 8 micrograms/rat) into substantia nigra. By subcutaneous administration, talipexole at 0.16 mg/kg and bromocriptine at 10.24 mg/kg induced significantly increased rotational behavior to the contralateral direction to the lesioned side. The onset of the effect was 30 min for talipexole and 90 min for bromocriptine. By intragastric administration, talipexole at 0.4 mg/kg and bromocriptine at 20.48 mg/kg significantly increased the rotational behavior, and the onset of the effect was 60 min for talipexole and 180 min for bromocriptine. Rotational behavior induced by talipexole was suppressed by a D2 antagonist, sulpiride (40 mg/kg, s.c.), but not by a D1 antagonist, SCH23390 (1 mg/kg, s.c.). In contrast, rotational behavior induced by bromocriptine was suppressed by both sulpiride and SCH23390. These results indicated that when the nigrostriatal dopaminergic functions are disrupted, talipexole stimulates the striatal postsynaptic dopamine receptors at much lower doses than bromocriptine. Also it was indicated that the stimulating effect of talipexole is solely mediated by dopamine D2 receptors, whereas the effect of bromocriptine is mediated by both D1 and D2 receptors.