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Biomedical subjects

I Spector

Publications and source records attributed to I Spector.

At least 19 recordsLinked to original sources
PubMed1997

Misakinolide A is a marine macrolide that caps but does not sever filamentous actin.

We have investigated the biochemical properties of the marine natural product, misakinolide A, a 40-membered dimeric lactone macrolide that differs from swinholide A only in the size of the macrolide ring. Analytical ultracentrifugation and steady-state fluorescence experiments show that misakinolide A binds simultaneously to two actin subunits with virtually the same affinity as swinholide A, suggesting that the modification in the ring size does not change the actin-binding site. Sedimentation equilibrium experiments suggest that binding is independent at each binding site, with a Kd of approximately 50 nM. Remarkably, misakinolide A does not sever actin filaments like swinholide A; rather, it caps the barbed end of F-actin. When capped by misakinolide A, the elongation rate constant at the barbed end is reduced to zero; pointed end growth was affected only to the extent that the compound sequesters unpolymerized actin. Misakinolide A has essentially no effect on the off-rate of actin subunits leaving the barbed end. Energy-minimized models of misakinolide A and swinholide A are consistent with conservation of identical binding sites in both molecules, but a difference in orientation of one binding site relative to the other may explain why swinholide A has severing activity whereas misakinolide A only has capping activity.

Actins↗
PubMed1996

Microfilament-disrupting agent latrunculin A induces and increased number of fenestrae in rat liver sinusoidal endothelial cells: comparison with cytochalasin B.

This report describes the effect of the actin-disrupting marine toxin latrunculin A on the cytoskeleton and fenestrae of liver endothelial cells (LECs). Fluorescence microscopy and whole mount-transmission electron microscopic preparations of isolated, purified, and cultured LECs showed that latrunculin A, which sequesters actin monomers and depolymerizes actin filaments, caused profound changes in microfilament organization in LECs. Scanning electron microscopic preparations showed that latrunculin A almost doubles the number of fenestrae within 10 minutes, whereas the diameter is only slightly reduced. All new fenestrae possess the earlier described fenestrae-associated cytoskeleton ring. Cytochalasin B, which disrupts the network of actin filaments, principally by capping the fast growing end of actin filaments, produced comparable effects with regard to actin organization and the number and size of fenestrae. After 1 hour of treatment, an equal maximum number of fenestrae was observed for both agents. The effect of latrunculin A was obtained at concentrations about 100 times lower than cytochalasin B. Thus, two agents that alter the state of actin organization in LECs, albeit by different mechanisms, cause the doubling of the number of fenestrae within 10 to 30 minutes. This indicates that the state of assembly of the actin cytoskeleton is important in the numerical dynamics of LEC fenestrae and that the actin cytoskeleton of LECs is probably the main mechanical regulator for sieving between the sinusoidal blood and the parenchymal cells. Latrunculin A represents a new agent in the study of the de novo formation of fenestrae.

Actin Cytoskeleton↗
PubMed1995

Swinholide A is a microfilament disrupting marine toxin that stabilizes actin dimers and severs actin filaments.

Swinholide A, isolated from the marien sponge Theonella swinhoei, is a 44-carbon ring dimeric dilactone macrolide with a 2-fold axis of symmetry. Recent studies have elucidated its unusual structure and shown that it has potent cytotoxic activity. We now report that swinholide A disrupts the actin cytoskeleton of cells grown in culture, sequesters actin dimers in vitro in both polymerizing and non-polymerizing buffers with a binding stoichiometry of one swinholide A molecule per actin dimer, and rapidly severs F-actin in vitro with high cooperativity. These unique properties are sufficient to explain the cytotoxicity of swinholide A. They also suggest that swinholide A might be a model for studies of the mechanism of action of F-actin severing proteins and be therapeutically useful in conditions where filamentous actin contributes to pathologically high viscosities.

3T3 Cells↗
PubMed1995

The state of actin assembly regulates actin and vinculin expression by a feedback loop.

Actin filaments are major determinants of cell shape, motility and adhesion, which control important biological processes including embryonic development and wound healing. These processes are associated with changes in actin assembly, which is regulated by controlling the balance between polymerized and non-polymerized actin. To maintain a significant pool of non-polymerized actin, mechanism(s) linking actin synthesis to its state of polymerization were proposed. We have studied this relationship between actin synthesis and organization by modulating actin assembly using different drugs. Unassembled actin was increased in 3T3 cells using either the Clostridium botulinum C2 toxin, which ADP-ribosylates actin, or by latrunculin A, a Red Sea sponge product, which binds monomeric actin. The synthesis of actin was dramatically reduced in these cells owing to a concomitant decrease in actin RNA level. Similar results were obtained with HeLa cells grown in both monolayer and in suspension, suggesting that cell shape changes associated with drug treatment are not the primary cause for the effect on actin synthesis. In contrast, the scrape-loading of 3T3 cells with phalloidin, a stabilizer of polymerized actin that increased the level of assembled actin, resulted in elevated actin synthesis and RNA content. The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, which is involved in actin-membrane associations, was altered in parallel with that of actin in cells treated with these drugs. The decrease in actin RNA resulted from destabilization of actin mRNA in cells where unassembled actin level was elevated. This is suggested by the unchanged transcription of actin in isolated nuclei from drug-treated cells, and by demonstrating that actin mRNA was degraded faster in cells after C2 toxin treatment than in control cells. This feedback control mechanism is mainly confined to the cytoplasm, as it remained active in enucleated cells. The results suggest the existence of an autoregulatory pathway for the expression of actin and other microfilament-associated proteins which is linked to the state of actin polymerization in the cell.

3T3 Cells↗
PubMed1993

Novel marine alkaloids from the tunicate Eudistoma sp. are potent regulators of cellular growth and differentiation and affect cAMP-mediated processes.

Six novel alkaloids that contain a fused tetracyclic pyrido[2,3,4-kl]acridine ring system were purified recently from the Red Sea purple tunicate Eudistoma sp. Evaluation of the effects of these alkaloids on cultured neuroblastoma and fibroblast cells revealed that they possess potent growth regulatory properties, and affect cell shape and adhesion. In mouse neuroblastoma cells, the Eudistoma alkaloids inhibited cell proliferation and induced a process of differentiation during which the cells flattened onto the surface, increased considerably in size, and extended long neurites. In hamster fibroblasts the alkaloids slowed down cell multiplication, and caused an exceptional cell flattening or elongation. In a virus-transformed derivative of the hamster fibroblasts the alkaloids restored many aspects of normal cell growth and morphology. In addition, several of the alkaloids mimicked the effects of cAMP analogs on two well-characterized cAMP-mediated processes involved in hepatic glucose metabolism--inhibition of pyruvate kinase (PK) activity and induction of mRNA for phosphoenolpyruvate carboxykinase (PEPCK). All these effects suggest that the Eudistoma alkaloids may act on the cAMP signaling system. However, a single application of these compounds was sufficient to completely block cell multiplication and to induce and sustain differentiation and "reverse transformation". Furthermore, these effects were not readily reversible following removal of the drugs. In contrast, a single application of agents that mimic or elevate cAMP induced a transient response that waned with time in culture, and the effects induced by constant elevation of cAMP reverse rapidly following drug removal. We propose that the Eudistoma alkaloids cause growth inhibition, differentiation, and reverse transformation by modifying the activity state of proteins that are involved in the regulation of cell shape and adhesion and serve as a target for the cAMP and/or other second messenger systems.

Alkaloids↗
PubMed1992

Intracellular cyclic AMP produces effects opposite to those of cyclic GMP and calcium on shape and motility of neuroblastoma cells.

We have directly evaluated the effects of various intracellular second messengers including cyclic nucleotides, calcium ion, and inositol polyphosphates on shape and motility of differentiating mouse neuroblastoma cells. The messengers were microinjected into cells and the responses of the soma, neurite, and growth cone were monitored using time-lapse video microscopy. Each messenger altered cell shape and motility in a characteristic manner. Cyclic AMP promoted lamellipodial expansion, neurite outgrowth, and motility. The other injected messengers opposed motility. Cyclic GMP caused motile structures to freeze and to retract permanently, while the inhibitory effects of calcium injection were concentration-dependent. Small calcium injections affected specifically actin-containing motile structures which froze and retracted temporarily. Intermediate calcium injections caused a strong contraction at the site of injection in all cells. With large injections, cells retracted long neurites, rounded up, and frequently began vigorous blebbing that continued to cell death. Injections of the inositol polyphosphates IP3(1,4,5) and IP4(1,4,5,6) mimicked the effects of small calcium injections, as did electrical stimulation that elicited action potentials. The results suggest that in mouse neuroblastoma cells, intracellular cAMP elevation increases cytoskeletal organization and promotes neurite extension perhaps through an enhancement of cell-substratum adhesion. On the other hand, a rise of intracellular cGMP or intracellular calcium interferes directly with the function and organization of the actin-microfilament system. The integrated action of these second messenger systems may, therefore, operate in vivo to allow substances released from neighboring cells to regulate neuronal architecture.

Bucladesine↗
PubMed1989

Beta-thalassaemia--the Johannesburg experience.

Twenty-nine patients with beta-thalassaemia major were treated in two Johannesburg hospitals between 1979 and 1984. They belonged to two ethnic groups--Mediterranean and Asian--and 53% were under the age of 6 years, the oldest being 20 years. Eight patients underwent splenectomy. All were regularly transfused and their quality of life greatly improved. The complication of regular transfusions is iron overload, which contributes to morbidity and mortality. Despite adequate iron excretion as a result of subcutaneous desferrioxamine (DFO) infusions, the patients showed significant iron overload, which suggested poor compliance in the home use of DFO and was the main long-term problem in the treatment of beta-thalassaemia major. Education needs to be directed at communities at risk and their doctors so that new cases can be prevented and the financial and emotional cost eased. If patients are given full support in complying with therapy, their life expectancy should be greatly increased.

Adolescent↗
PubMed1989

Are regular 'top-up' transfusions necessary in otherwise well, growing premature infants?

To test the hypothesis that very low-birth-weight neonates who are otherwise well and are growing, are not compromised if their haemoglobin concentration is allowed to fall below 10 g/dl, 20 such neonates were randomly divided into two groups. The infants in one group received 'top-up' transfusions to keep their haemoglobin concentrations above 10 g/dl, while haemoglobin concentrations in the other group were allowed to fall below this level. Although the group which did not receive transfusions had significantly lower haemoglobin values, no significant differences in clinical well-being and daily mass gain were observed. The non-transfused group also had significantly higher reticulocyte counts. We conclude that it is not necessary to administer routine top-up transfusions to these infants.

Blood Transfusion↗
PubMed1989

Latrunculins--novel marine macrolides that disrupt microfilament organization and affect cell growth: I. Comparison with cytochalasin D.

The latrunculins are architecturally novel marine compounds isolated from the Red Sea sponge Latrunculia magnifica. In vivo, they alter cell shape, disrupt microfilament organization, and inhibit the microfilament-mediated processes of fertilization and early development. In vitro, latrunculin A was recently found to affect the polymerization of pure actin in a manner consistent with the formation of a 1:1 molar complex with G-actin. These in vitro effects as well as previous indications that the latrunculins are more potent than the cytochalasins suggest differences in the in vivo mode of action of the two classes of drugs. To elucidate these differences we have compared the short- and long-term effects of latrunculins on cell shape and actin organization to those of cytochalasin D. Exposure of hamster fibroblast NIL8 cells for 1-3 hr to latrunculin A, latrunculin B, and cytochalasin D causes concentration-dependent changes in cell shape and actin organization. However, the latrunculin-induced changes were strikingly different from those induced by cytochalasin D. Furthermore, while initial effects were manifest with both latrunculin A and cytochalasin D already at concentrations of about 0.03 microgram/ml, latrunculin A caused complete rounding up of all cells at 0.2 microgram/ml, whereas with cytochalasin D maximum contraction was reached at concentrations 10-20 times higher. The short-term effects of latrunculin B were similar to those of latrunculin A although latrunculin B was slightly less potent. All three drugs inhibited cytokinesis in synchronized cells, but their long-term effects were markedly different. NIL8 cells treated with latrunculin A maintained their altered state for extended periods. In contrast, the effects of cytochalasin D progressed with time in culture, and the latrunculin B-induced changes were transient in the continued presence of the drug. These transient effects were found to be due to a gradual inactivation of latrunculin B by serum and were used to compare recovery patterns of cell shape and actin organization in two different cell lines. This comparison showed that the transient effects of latrunculin B were fully reversible for the NIL8 cells and not for the mouse neuroblastoma N1E-115 cells.

Actin Cytoskeleton↗
PubMed1988

Client attributions for sexual dysfunction.

This investigation examined attributions for sexual dysfunctions made by 63 individuals and 21 of their partners who presented at a sex therapy service for the following problems: erectile dysfunction, premature ejaculation, and female orgasmic dysfunctions. All participants completed measures of marital adjustment, locus of control, depression and a questionnaire which assessed: attributions of responsibility for the sexual problem, perceived control over sexual functioning, distress, effort made to improve the sexual relationship, and expectations about the efficacy of sex therapy for the problem. Results indicate that both identified patients and their partners, regardless of the dysfunction, blamed the sexual problem on the "dysfunctional individual" rather than on the circumstances or the partner. With respect to the partners, husbands of women with orgasmic dysfunction were more likely to blame themselves than the circumstances, while the opposite was true for wives of males with erectile difficulties. Individuals experiencing the dysfunction perceived themselves and their partners as having little, but equal control over the identified patient's sexuality. Correlational analyses indicate that in identified patients, the better the quality of the marital relationship, the greater the self-blame and the lower the partner blame. Those with happy marriages also made greater efforts to improve their sexual relationship and had higher expectations of success with therapy. The implications of the results for research on the role of attributions in sexual dysfunction and for assessment of cognitive factors in sexually dysfunctional individuals and their partners is discussed.

Adult↗
PubMed1987

Malignant fibrous histiocytoma of the breast. A case report.

Primary malignant fibrous histiocytoma of the breast was diagnosed in a 45-year-old Indian woman. She presented with a large ulcerated lesion and had lung metastases. Treatment was by radiotherapy, toilet mastectomy and chemotherapy. The patient died of rapid extension of lung metastases 15 months after first being seen. Although this tumour has previously been considered to have a relatively good prognosis, this report emphasises its fully malignant metastatic potential.

Breast Neoplasms↗
PubMed1987

Acute mixed-lineage leukaemia involving myeloid and T-cell phenotypes. A case report.

Among the rare acute mixed-lineage leukaemias, a myeloid T-cell phenotype has recently been recognised. An additional case characterised by myeloid cytochemistry, development of Auer rods in culture and Tdt, WT1 and T11 phenotypes on cell-marker analysis is reported. The patient died from disseminated non-reactive tuberculosis after chemotherapy.

Adolescent↗
PubMed1987

Inhibition of actin polymerization by latrunculin A.

Latrunculin A, a toxin purified from the red sea sponge Latrunculia magnifica, was found previously to induce striking reversible changes in the morphology of mammalian cells in culture and to disrupt the organization of their microfilaments. We now provide evidence that latrunculin A affects the polymerization of pure actin in vitro in a manner consistent with the formation of a 1:1 molar complex between latrunculin A and G-actin. The equilibrium dissociation constant (Kd) for the reaction in vitro is about 0.2 microM whereas the effects of the drug on cultured cells are detectable at concentrations in the medium of 0.1-1 microM.

Actins↗
PubMed1987

A unique 8;16 translocation in two infants with poorly differentiated monoblastic leukemia.

Acute monoblastic leukemia is nonrandomly associated with abnormalities involving 11q. Two infants, one a neonate and the other 19 months of age, had the same hitherto undescribed karyotypic abnormality, t(8;16)(p11;p13), associated with acute nonlymphocytic leukemia M5a. The older child had an additional translocation, t(10;11)(q11;p15), but the chromosome arms affected were the opposite to those described in acute nonlymphocytic leukemia M5a of childhood. Therefore, it is postulated that genes involved in monocytic differentiation may be situated on 8p11 or 16p13, as well as on 11q.

Bone Marrow↗
PubMed1987

Malignant melanomas in the Eur-African-Malay population of South Africa.

Malignant melanoma is common among the whites of South Africa. In the black population, the tumor is much less frequent and occurs predominantly on the lower limb--particularly the sole of the foot. This study brings to light the anomalous situation that among the Eur-African-Malay population (those of mixed ancestry), malignant melanoma has probably the lowest incidence in the world.

Adult↗
PubMed1987

Development of sodium channel protein during chemically induced differentiation of neuroblastoma cells.

We have previously shown that the [3H]saxitoxin binding site of the sodium channel is expressed independently of the [125I]scorpion toxin binding site in chick muscle cultures and in rat brain. In the present work, we studied the development of the sodium channel protein during chemically induced differentiation of N1E-115 neuroblastoma cells, using [3H]saxitoxin binding, [125I]scorpion toxin binding, and 22Na uptake techniques. When grown in their normal culture medium, these cells are mostly undifferentiated, bind 90 +/- 10 fmol of [3H]saxitoxin/mg of protein and 112 +/- 14 fmol of [125I]scorpion toxin/mg protein, and, when stimulated with scorpion toxin and batrachotoxin, take up 70 +/- 5 nmol of 22Na/min/mg of protein. Cells treated with dimethyl sulfoxide (DMSO) or hexamethylene-bis-acetamide (HMBA) differentiate morphologically within 3 days. At this time, the [3H]saxitoxin binding, the [125I]scorpion toxin binding, and the 22Na uptake values are not very different from those of undifferentiated cells. With subsequent time in DMSO or HMBA, these values continue to increase, a result indicating that the main period of sodium channel expression occurs well after the cells have assumed the morphologically differentiated state. The data indicate that the expression of sodium channels and morphological differentiation are independently regulated neuronal properties, that the attainment of morphological differentiation is necessary but not in itself sufficient for full expression of the sodium channel proteins, and that, in contrast to the chick muscle cultures and rat brain, the [3H]saxitoxin site and [125I]scorpion toxin site appear to be coregulated in N1E-115 cells.

Acetamides↗