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Biomedical subjects

A Singer

Publications and source records attributed to A Singer.

At least 19 recordsLinked to original sources
PubMed2001

Perineuronal nets in the rat medial nucleus of the trapezoid body surround neurons immunoreactive for various amino acids, calcium-binding proteins and the potassium channel subunit Kv3.1b.

Perineuronal nets (PNs) are known as chondroitin sulfate-rich, lattice-like coatings of the extracellular matrix ensheathing mainly GABAergic, parvalbumin-containing neurons especially in the cerebral cortex. PNs have also been detected around GABA-immunonegative cells which were shown to be not aminergic, cholinergic, nitrinergic or peptidergic in various brain regions of some mammalian species. To find out whether glycine and aspartate may occur in net-bearing neurons the present study was focused on the rat medial nucleus of the trapezoid body (MNTB) which contains a large portion of cells immunoreactive for these amino acids, but appears to be devoid of GABA-immunoreactive cell bodies. PNs were detected around many glycine- and aspartate-immunopositive neurons in the MNTB by carbocyanine double labeling and confocal laser scanning microscopy. An additional finding was that the lectin-cytochemically stained extracellular matrix surrounds the calretinin-immunoreactive calyces of Held known as giant glutamatergic endbulbs which cover glycinergic principal cells in the MNTB. As elucidated by triple fluorescence labeling, the vast majority of somata co-expressed the calcium-binding proteins parvalbumin and calbindin, but not calretinin. The observed co-localization of PNs and immunoreactivity for the voltage-dependent potassium channel Kv3.1b - as an established marker of fast-firing parvalbumin-containing neurons - supports the assumed function of PNs as a cation exchanger ensuring rapid ion transport as required by highly active nerve cells.

Amino Acids↗
PubMed2001

PRAM-1 is a novel adaptor protein regulated by retinoic acid (RA) and promyelocytic leukemia (PML)-RA receptor alpha in acute promyelocytic leukemia cells.

The t(15;17) translocation, found in 95% of acute promyelocytic leukemia, encodes a promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARalpha) fusion protein. Complete remission of acute promyelocytic leukemia can be obtained by treating patients with all-trans retinoic acid, and PML-RARalpha plays a major role in mediating retinoic acid effects in leukemia cells. A main model proposed for acute promyelocytic leukemia is that PML-RARalpha exerts its oncogenic effects by repressing the expression of retinoic acid-inducible genes critical to myeloid differentiation. By applying subtraction cloning to acute promyelocytic leukemia cells, we identified a retinoic acid-induced gene, PRAM-1 (PML-RARalpha target gene encoding an Adaptor Molecule-1), which encodes a novel adaptor protein sharing structural homologies with the SLAP-130/fyb adaptor. PRAM-1 is expressed and regulated during normal human myelopoiesis. In U937 myeloid precursor cells, PRAM-1 expression is inhibited by expression of PML-RARalpha in the absence of ligand and de novo superinduced by retinoic acid. PRAM-1 associates with other adaptors, SLP-76 and SKAP-55HOM, in myeloid cell lines and with protein tyrosine kinase lyn. By providing the first evidence that PML-RARalpha dysregulates expression of an adaptor protein, our data open new insights into signaling events that are disrupted during transformation by PML-RARalpha and induced by retinoic acid during de novo differentiation of acute promyelocytic leukemia cells.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Maturation versus death of developing double-positive thymocytes reflects competing effects on Bcl-2 expression and can be regulated by the intensity of CD28 costimulation.

Immature double-positive (DP) thymocytes mature into CD4(+)CD8(-) cells in response to coengagement of TCR with any of a variety of cell surface "coinducer" receptors, including CD2. In contrast, DP thymocytes are signaled to undergo apoptosis by coengagement of TCR with CD28 costimulatory receptors, but the molecular basis for DP thymocyte apoptosis by TCR plus CD28 coengagement is not known. In the present study, we report that TCR plus CD28 coengagement does not invariably induce DP thymocyte apoptosis but, depending on the intensity of CD28 costimulation, can induce DP thymocyte maturation. We demonstrate that distinct but interacting signal transduction pathways mediate DP thymocyte maturation signals and DP thymocyte apoptotic signals. Specifically, DP maturation signals are transduced by the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and up-regulate expression of the antiapoptotic protein Bcl-2. In contrast, the apoptotic response stimulated by CD28 costimulatory signals is mediated by ERK/MAPK-independent pathways. Importantly, when TCR-activated thymocytes are simultaneously coengaged by both CD28 and CD2 receptors, CD28 signals can inhibit ERK/MAPK-dependent Bcl-2 protein up-regulation. Thus, there is cross-talk between the signal transduction pathways that transduce apoptotic and maturation responses, enabling CD28-initiated signal transduction pathways to both stimulate DP thymocyte apoptosis and also negatively regulate maturation responses initiated by TCR plus CD2 coengagement.

Animals↗
PubMed2001

Cervical cancer detection by hybrid capture and evaluation of local risk factors.

UNLABELLED: The city of Recife, northeastern Brazil, is reported to have the highest incidence of cervical cancer worldwide (83.2/100000 women). OBJECTIVE: To evaluate the efficacy of Hybrid Capture I (HC I) in cervical cancer detection and some risk factors in Recife. METHOD: Cervical scrapes for HC I analysis followed by colposcopy were collected from 140 women (70 with cervical cancer and 70 with normal cervix) from three screening services in Recife. RESULT: HC I sensitivity and specificity were 82.9 and 41.4%, respectively. The odds ratios for cervical cancer when Gesta > or = 5 and vaginal parity > or = 4 were, respectively, 5.30 and 4.27. CONCLUSION: HC I is a moderately sensitive method to detect cervical cancer, but it does not seem to be useful as a primary screening tool for it's low specificity. Early pregnancy, high Gesta/Para and living in rural areas were important local risk factors.

Adult↗
PubMed2001

Strength of signaling by CD4 and CD8 coreceptor tails determines the number but not the lineage direction of positively selected thymocytes.

The present study has assessed the impact of the intracellular domains of CD4 and CD8 on positive selection and lineage direction of MHC class I-restricted thymocytes. Contrary to current presumption, we found that the CD4 tail promotes the generation of both CD4+ and CD8+ T cells without preference for the CD4+ T cell lineage. We also found that the identity of the coreceptor tail and hence the strength of coreceptor signaling determine the number of thymocytes undergoing positive selection but not their ultimate CD4/CD8 phenotype. These findings demonstrate that the strength of coreceptor signaling has a significant quantitative but not qualitative impact on positive selection and provide a simple explanation for the greater numbers of CD4+ than CD8+ T cells selected in the normal thymus.

Animals↗
PubMed2001

Identification of nucleated cells in urine using lectin staining.

Microscopic examination of urinary sediment is an integral component in the evaluation of nephropathies. However, identification and differentiation of the nucleated nonsquamous cells in urine is often difficult using such conventional techniques as phase contrast or bright field microscopy, even after Papanicolaou staining, and requires a lot of experience. We now report a method to differentiate urinary cell types using lectin staining. Twenty-five lectins were examined with respect to their binding pattern on cryosections of the human kidney and urinary tract, as well as binding to blood cells. The specificity of lectin binding to a cell type both in situ and in urine was confirmed by double labeling with specific antibodies directed against various sections of the nephron or nucleated blood cells. For urine cytologic examinations, acetone-fixed cytopreparations of urinary sediments were incubated with a combination of a fluorescein isothiocyanate (FITC)-coupled and a rhodamine-coupled lectin, followed by staining of the nuclei with 4',6-diamidino-2-phenylindole. Specimens were examined in triple immunofluorescence (FITC/rhodamine/UV). Cell types could be identified by their characteristic lectin-binding pattern. For example, the lectin combination of Sophora japonica agglutinin (aggl; SJA) and Erythrina cristagalli aggl (ECA) permitted a differentiation between cells of the proximal tubules (SJA positive [SJA+], ECA+), distal tubules (SJA negative [SJA-], ECA+), collecting ducts (SJA+, ECA-), and lymphocytes (SJA-, ECA-). In preliminary studies, examination of urinary sediment in various chronic nephropathies by this technique showed differences in their cellular excretion pattern. In summary, staining urinary sediments with combinations of lectins provides a rapid and relatively inexpensive method for a facilitated and reliable differentiation of the various nucleated cell types in urine.

Binding Sites↗
PubMed2001

Evaluation of synaptosomal uptake inhibition of most relevant constituents of St. John's wort.

In our previous investigations, we could demonstrate that extract preparations of Hypericum perforatum (St. John's wort, SJW) inhibit the uptake of several neurotransmitters (serotonin, norepinephrine, dopamine, GABA, L-glutamate) in synaptosomal preparations of rodent brain. Hyperforin, the lipophilic constituent, was identified as the main component responsible for these effects. The properties seen for hyperforin in these and other pharmacological models present a plausible and logical explanation for the well documented antidepressive effects of SJW extract preparations in clinical studies. However, evidence for other active principles in SJW extract have been reported (See also communications by Misane & Ogren and Philippu in this issue). Accordingly, we tested various SJW extract preparations and all relevant constituents as possible inhibitors of synaptosomal uptake of neurotransmitters. Two further components were found to be active in those models. Adhyperforin, like hyperforin, showed a strong inhibiting profile in all uptake systems investigated. Moreover, we could observe a weak to moderate inhibiting profile for the oligomeric procyanidins fraction (OPC). Further investigations would have to clarify any possible contribution of these two constituents to the antidepressive effects of SJW extract seen in animal experiments and clinical trials.

Animals↗
PubMed2001

Hyperforin--antidepressant activity by a novel mechanism of action.

Hyperforin represents a major antidepressive constituent of St. John's wort (SJW) extract. It not only inhibits the neuronal uptake of serotonin, norepinephrine and dopamine like many other antidepressants, but also inhibits GABA and L-glutamate uptake. This broad-spectrum effect is obtained by an elevation of the intracellular Na+ concentration, probably due to activation of sodium conductive pathways not yet finally identified but most likely ionic channels. This makes hyperforin the first member of a new class of compounds with a preclinical antidepressant profile due to a completely novel mechanism of action.

Animals↗
PubMed2001

The effects of octylcyanoacrylate on scarring after burns.

OBJECTIVE: To compare the effects of octylcyanoacrylate (OCA), silver sulfadiazine (SSD), polyurethane film (PU), and dry gauze (G) on scarring three months after partial-thickness burns. METHODS: This was a prospective, blinded, controlled experimental trial using isoflurane-anesthetized swine. Standardized partial-thickness burns were inflicted by applying an aluminum bar preheated to 80 degrees C to the backs and flanks of a young pig for 20 seconds. Four equal sets of ten burns each were randomly treated with OCA spray, SSD, PU, or G. Dressing changes were performed on days 1, 2, 3, and 4 after injury. Digital images of the burns were obtained immediately and three months later for masked computerized determination of scar surface area. Full-thickness biopsies were taken at three months for masked histopathological evaluation. The primary outcome was the percent reduction in residual wound area (RWA) calculated by subtracting the area of each individual burn from the area of the largest burn and dividing this value by the area of the largest burn (intraobserver correlation, r = 0.99). Secondary outcomes were the proportion of burns with the presence of scar tissue (abnormal collagen under polarized light; intraobserver agreement, kappa = 0.93) and the cosmetic appearance on a 100-mm visual analog scale marked "best scar" at the high end (inter-observer correlation, r = 0.82). Analysis of variance (ANOVA) and chi(2) tests were used for group comparisons as appropriate. This study had 80% power to detect a 33-percentage-point difference in RWA among groups (alpha = 0.05). RESULTS: A total of 40 burns were inflicted on the pig. There was no difference in percent RWA across the groups (OCA = 25%, SSD = 40%, PU = 25%, G = 32%; p = 0.13). There was no difference in the proportion of wounds with scarring among the groups (OCA = 10%, SSD = 22%, PU = 2%, G = 30%; p = 0.89). There was also no difference in the cosmetic scores among the groups (OCA = 78 mm, SSD = 75 mm, PU = 74 mm, G = 74 mm; p = 0.96). CONCLUSIONS: The effects of OCA spray, SSD, PU, and dry gauze on scarring three months after burns in pigs are similar.

Animals↗
PubMed2000

[Mechanism of action of St. Johns wort extract].

Over the last few years St-John's wort preparations have been used in large quantities in Germany for treating mild to moderate depression. In the meantime the antidepressive action of these extracts has been proved in numerous placebo-controlled studies. Furthermore, a considerably lower adverse effect rate compared with classic antidepressants has been established. Analogously to other antidepressants, subchronic St-John's wort treatment of rats showed significant down-regulation of beta receptor density and a significant increase in 5HT2 receptors. The extract also exhibited antidepressant activity in animal pharmacological models of depression. Interest is now focused on identifying the underlying pharmacological mechanisms of action of this phytotherapeutic agent. Like other working parties, we were only able to identify a weak inhibitory effect of the extract and the pure substance hypericin on the monoamine oxidases A and B. Similarly to synthetic antidepressants, St-John's wort exerts marked inhibitory effects on synaptosomal uptake of serotonin and noradrenaline. However, dopamine and uptake and neuronal uptake of GABA and L-glutamate are also inhibited. These effects may mainly be attributed to the substance hyperforin contained in the extract. An additional, as yet unknown, pharmacological mechanism of action of St-John's wort extracts is beginning to emerge. Although hyperforin shows similar properties to classical antidepressants, there are indications of a novel mechanism of action. Our laboratory is currently investigating the means by which St-John's wort extract, or its constituent hyperforin, develops its antidepressant action.

Animals↗
PubMed2000

Distinct arachidonate-releasing functions of mammalian secreted phospholipase A2s in human embryonic kidney 293 and rat mastocytoma RBL-2H3 cells through heparan sulfate shuttling and external plasma membrane mechanisms.

We analyzed the ability of a diverse set of mammalian secreted phospholipase A(2) (sPLA(2)) to release arachidonate for lipid mediator generation in two transfected cell lines. In human embryonic kidney 293 cells, the heparin-binding enzymes sPLA(2)-IIA, -IID, and -V promote stimulus-dependent arachidonic acid release and prostaglandin E(2) production in a manner dependent on the heparan sulfate proteoglycan glypican. In contrast, sPLA(2)-IB, -IIC, and -IIE, which bind weakly or not at all to heparanoids, fail to elicit arachidonate release, and addition of a heparin binding site to sPLA(2)-IIC allows it to release arachidonate. Heparin nonbinding sPLA(2)-X liberates arachidonic acid most likely from the phosphatidylcholine-rich outer plasma membrane in a glypican-independent manner. In rat mastocytoma RBL-2H3 cells that lack glypican, sPLA(2)-V and -X, which are unique among sPLA(2)s in being able to hydrolyze phosphatidylcholine-rich membranes, act most likely on the extracellular face of the plasma membrane to markedly augment IgE-dependent immediate production of leukotriene C(4) and platelet-activating factor. sPLA(2)-IB, -IIA, -IIC, -IID, and -IIE exert minimal effects in RBL-2H3 cells. These results are also supported by studies with sPLA(2) mutants and immunocytostaining and reveal that sPLA(2)-dependent lipid mediator generation occur by distinct (heparanoid-dependent and -independent) mechanisms in HEK293 and RBL-2H3 cells.

Amino Acid Sequence↗
PubMed2000

Immature thymocytes undergoing receptor rearrangements are resistant to an Atm-dependent death pathway activated in mature T cells by double-stranded DNA breaks.

Immature CD4(+)CD8(+) thymocytes rearrange their T cell receptor (TCR)-alpha gene locus to generate clonotypic alpha/beta TCR, after which a few cells expressing selectable TCR are signaled to further differentiate into mature T cells. Because of requirements for self-tolerance, immature CD4(+)CD8(+) thymocytes are programmed to die in the thymus in response to a variety of stimuli that do not induce death of mature T cells. We now demonstrate that, in contrast to all previously described stimuli, immature CD4(+)CD8(+) thymocytes are selectively more resistant than mature T cells to apoptotic death induced by DNA intercalating agents. Importantly, we demonstrate that DNA intercalating agents induce double-stranded DNA breaks in both immature thymocytes and mature T cells, but immature thymocytes tolerate these DNA breaks, whereas mature T cells are signaled to die by an Atm-dependent but p53-independent death mechanism. Thus, our results indicate that absence of an Atm-dependent but p53-independent pathway allows immature thymocytes to survive double-stranded DNA breaks. It is likely that the unique ability of immature thymocytes to survive DNA-damaging intercalating agents reflects their tolerance of double-stranded DNA breaks that occur normally during antigen receptor gene rearrangements.

Animals↗