Dynamin inhibits phosphatidylinositol 3-kinase in hematopoietic cells.

Phosphatidylinositol 3-kinase (PI 3-kinase) plays a role in late stages of endocytosis as well as in cellular proliferation and transformation. The SH3 domain of its regulatory p85 subunit stimulates the GTPase activity of dynamin in vitro. Dynamin is a GTPase enzyme required for endocytosis of activated growth factor receptors. An interaction between these proteins has not been demonstrated in vivo. Here, we report that dynamin associates with PI 3-kinase in hematopoietic cells. We detected both p85 and PI 3-kinase activity in dynamin immune complexes from IL-3-dependent BaF3 cells. However, this association was significantly reduced in BaF3 cells transformed with the BCR/abl oncogene. After transformation only a 4-fold increase in PI 3-kinase activity was detected in dynamin immune complexes, whereas grb2 associated activity was elevated 20-fold. Furthermore, dynamin inhibited the activity of both purified recombinant and immunoprecipitated PI 3-kinase. In BaF3 cells expressing a temperature-sensitive mutant of BCR/abl, a significant decrease in p85 and dynamin association was observed 4 h after the induction of BCR/abl activity. In contrast, in IL-3-stimulated parental BaF3 cells, this association was increased. Our results demonstrate an in vivo association of PI 3-kinase with dynamin and this interaction regulates the activity of PI 3-kinase.

Does strategy knowledge influence working memory in children with mathematical disabilities?

This study investigated the relationship between working memory (WM), declarative strategy knowledge, and math achievement in children with and without mathematical disabilities (MD). Experiment 1 examined the relationship between strategy knowledge, verbal WM, and visual-spatial WM in children with MD as a function of initial, gain, and maintenance conditions. The results showed that after partialing the influence of reading, stable strategy choices rather than specific strategy knowledge was related to verbal and visual-spatial WM span in high demand (maintenance) conditions. Experiment 2 compared children with MD to a group of chronological age-matched children and a group of math ability-matched children on the same conditions as Experiment 1. Age-matched children's verbal and visual-spatial WM performance was superior to that of children with MD, whereas WM performance was statistically comparable between children with MD and younger children matched on math ability. The selection of expert strategies was related to high WM span scores in the initial conditions. After controlling for reading achievement in a regression analysis, verbal and visual-spatial WM, stable verbal strategy choices, and expert strategy choices related to visual-spatial processing all contributed independent variance to math achievement. Overall, these results suggest that WM and math achievement are related to strategy knowledge.

Serum markers of lamellar basement membrane degradation and lamellar histopathological changes in horses affected with laminitis.

In order better to evaluate the extent to which degradation of the lamellar basement membrane (LBM) by matrix metalloproteinases (MMP) occurs in equine laminitis, we determined the concentration of type IV collagen and laminin in normal and laminitic horses, using specific immunoassays. Blood samples were obtained from both the jugular and the cephalic veins of horses (n = 10) before and after the induction of acute alimentary laminitis by carbohydrate overload. Jugular and cephalic venous blood samples were also obtained from horses affected with naturally occurring laminitis (n = 16) and nonlaminitic controls (n = 8). The serum collagen IV concentration was not changed following the induction of laminitis in the experimental group. Serum collagen IV concentration was increased in jugular venous blood obtained from cases of naturally occurring laminitis (mean +/- s.e. 218.04 +/- 18.59 ng/ml) compared with nonlaminitic controls (157.50 +/- 10.93 ng/ml) (P<0.05). Serum collagen IV concentration was also increased in jugular venous blood obtained from severely laminitic horses (219.50 +/- 18.18 ng/ml) compared with nonlaminitic controls (157.50 +/- 10.93 ng/ml) (P<0.05). A difference in serum concentration of collagen IV was not identified based on chronicity of naturally occurring laminitis. Serum laminin concentration did not differ between laminitic and nonlaminitic horses. Differences in serum laminin concentration were not identified based on sampling location (jugular or cephalic vein), severity of laminitic pain, or chronicity of spontaneous laminitis. In conclusion, the circulating concentration of collagen IV was increased in horses affected with naturally occurring laminitis. The potential role for serum collagen IV assay for characterisation of equine laminitis warrants further investigation.

PI 3-kinase activation in BCR/abl-transformed hematopoietic cells does not require interaction of p85 SH2 domains with p210 BCR/abl.

BCR/abl is a chimeric oncogene implicated in the pathogenesis of human chronic myelogenous leukemia. Expression of the BCR/abl gene induces hematologic malignancies in transgenic mice and transformation of interleukin-3-dependent hematopoietic cells. The mechanism of BCR/abl-mediated transformation of hematopoietic cells is poorly understood and involves activation of at least two signaling pathways, p21ras and PI 3-kinase. Here we report that PI 3,4-P2 and PI 3,4,5-P3, the enzymatic products of PI 3-kinase, accumulate in metabolically labeled transformed hematopoietic cells, in contrast to our previous report on the lack of accumulation of PI 3-kinase products in nontransformed NIH 3T3 fibroblasts that express p210 BCR/abl. Transformed cells also have increased PI 3-kinase activity in total cell extracts and membrane fractions. Activation of PI 3-kinase occurs by occupancy of SH2 domains of PI 3-kinase regulatory subunit, p85, by phosphorylated YXXM motifs. Therefore, we investigated whether BCR/abl binds to p85 and whether this binding is mediated by interaction of p85 SH2 domains with YXXM motif of BCR/abl. Association of p210 BCR/abl with p85 in immune complexes and with p85 SH2 domains was evident in hematopoietic cells that express the wt p210 BCR/abl. However, the binding of BCR/abl to p85 SH2 domains was abolished in cells expressing mutant, temperature-sensitive (ts) p210 BCR/abl in which the tyrosine in the YXXM motif of p210 BCR/abl was replaced by histidine. Despite lack of direct interaction with p85 SH2 domains, expression of ts p210 BCR/abl resulted in rapid, time-dependent activation of total and membrane-associated PI 3-kinase and increased PI 3-kinase activity in anti-P-tyr and anti-abl immunoprecipitates. These data suggest that BCR/abl-induced activation of PI 3-kinase in hematopoietic cells does not require binding of p85 SH2 domains to BCR/abl gene product and involves interaction with other tyrosine phosphorylated intermediate proteins.

Serine-rich region of the IL-2 receptor beta-chain is required for activation of phosphatidylinositol 3-kinase.

The intracellular portion of the IL-2 receptor (IL-2R) signal transducing beta-chain contains a distinct region, designated "serine-rich," which encompasses sequences required for IL-2-mediated cell growth. Although the receptor does not possess intrinsic protein-tyrosine kinase activity, IL-2 binding induces activation of intracellular protein-tyrosine kinases. Activation of many protein-tyrosine kinases leads to activation of phosphatidylinositol 3-kinase (PI 3-kinase). IL-2 binding also induces activation of PI 3-kinase. To study the interaction of PI 3-kinase with the IL-2 receptor beta-chain we analyzed PI 3-kinase activity in cells which express the wild type and mutant beta-chain. IL-2 mediated an increase in association with PI 3-kinase activity and protein in immunoprecipitates from cells expressing mitogenically competent receptors. PI 3-kinase products also increased in response to IL-2 in these cells. Deletion of the beta-chain serine-rich region abolished IL-2-mediated mitogenesis and cells expressing this mutant failed to activate PI 3-kinase. The interaction of the IL-2 receptor with an intracellular tyrosine kinase, lck, has been mapped to the acidic-rich region of the beta-chain. Cells which express the beta-chain lacking the acidic-rich region grow in the presence of IL-2 and had IL-2-dependent activation of PI 3-kinase. Activation of PI 3-kinase in response to IL-2 was not abolished by treatment of cells with rapamicin and occurred only in cells which express mitogenically competent receptors. The results presented in this study suggest that IL-2-mediated PI 3-kinase activation occurs by a mechanism distinct from interaction with the lck protein-tyrosine kinase.

Platelet-derived growth factor activates membrane-associated phosphatidylinositol 3-kinase and mediates its translocation from the cytosol. Detection of enzyme activity in detergent-solubilized cell extracts.

Phosphatidylinositol 3-kinase (PI 3-kinase) activity has been detected in immune complexes with active protein tyrosine kinases, and its products have been measured in intact cells in response to growth stimuli. Both methods do not directly evaluate whole cell PI 3-kinase enzymatic activity. We have developed a sensitive method to measure PI 3-kinase activity in diluted, detergent-containing whole cell extracts and used this method to determine total, soluble, and membrane-associated PI 3-kinase activity in PDGF-stimulated NIH 3T3 fibroblasts. PDGF stimulation induced a 1.4-fold increase in total Nonidet P-40-extractable PI 3-kinase activity, which occurred within 1 min and was maintained above basal levels at 10 min. At the same time, PI 3-kinase activity in the soluble fraction decreased 30-50%. However, membrane-bound PI 3-kinase activity increased 2.4-fold at 1 min and 3.1-fold at 5 min. Translocation of the p85 PI 3-kinase subunit to the membrane was maximal at 10 min. These results suggest that PDGF-mediated activation of PI 3-kinase in membrane fraction results from initial intrinsic enzymatic activation followed by translocation from the cytosol.

Human parvovirus B-19: not just a pediatric problem.

Parvoviruses have long been associated with disabling and even fatal illnesses in animals. The discovery of the human parvovirus B-19 in 1975 (1) and subsequent studies of its effects in humans identified this virus as the causative agent of erythema infectiosum ("fifth disease") in children. (2). Erythema infectiosum (EI) is a common, self-limited infectious disorder in children, easily recognized by the classic "slapped cheek" facial erythema and fine reticular rash. Only in the 1980s have further investigations linked HPV B-19 infection with more significant clinical syndromes, among which is an adult polyarthropathy. This presentation in adults is more common than is currently understood and is easily confused with other symmetric polyarthropathies. Recognition and conservative treatment of this disorder are important for the emergency physician, to whom these patients may present.

Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate.

The generation of second messengers from the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdInsP2) by phosphoinositidase C has been implicated in the mediation of cellular responses to a variety of growth factors and oncogene products. The first step in the production of PtdInsP2 from phosphatidylinositol (PtdIns) is catalysed by PtdIns kinase. A PtdIns kinase activity has been found to associate specifically with several oncogene products, as well as with the platelet-derived growth factor (PDGF) receptor. We have previously identified two biochemically distinct PtdIns kinases in fibroblasts, and have found that only one of these, designated type I, specifically associates with activated tyrosine kinases. We have now characterized the site on the inositol ring phosphorylated by type I PtdIns kinase, and find that this kinase specifically phosphorylates the D-3 ring position to generate a novel phospholipid, phosphatidylinositol-3-phosphate (PtdIns(3)P). In contrast, the main PtdIns kinase in fibroblasts, designated type II, specifically phosphorylates the D-4 position to produce phosphatidylinositol-4-phosphate (PtdIns(4)P), previously considered to be the only form of PtdInsP. We have also tentatively identified PtdIns(3)P as a minor component of total PtdInsP in intact fibroblasts. We propose that type I PtdIns kinase is responsible for the generation of PtdIns(3)P in intact cells, and that this novel phosphoinositide could be important in the transduction of mitogenic and oncogenic signals.

Nitrogen catabolite repression in a glutamate auxotroph of Saccharomyces cerevisiae.

The biosynthesis of asparaginase II in Saccharomyces cerevisiae is subject to nitrogen catabolite repression. In the present study we examined the physiological effects of glutamate auxotrophy on cellular metabolism and on the nitrogen catabolite repression of asparaginase II. Glutamate auxotrophic cells, incubated without a glutamate supplement, had a diminished internal pool of alpha-ketoglutarate and a concomitant inability to equilibrate ammonium ion with alpha-amino nitrogen. In the glutamate auxotroph, asparaginase II biosynthesis exhibited a decreased sensitivity to nitrogen catabolite repression by ammonium ion but normal sensitivity to nitrogen catabolite repression by all amino acids tested.

The effect of lithium carbonate on alcoholism in 20 male patients with concurrent major affective disorder.

Descriptively, Alcoholism and Bipolar Affective Disorders may resemble each other. Both disorders are marked by a chronic course with periods of remission and relapse and often profound affective disturbance. Since lithium carbonate has shown a remarkable efficacy in the treatment or prophylaxis of Bipolar Affective Disorder, it would be natural to hope for some similar dramatic benefit in alcoholism. Two controlled studies have been published in English in which lithium salts showed some benefits on drinking in "depressed" patients with alcoholism. Other studies published in the literature have been less certain but cautiously positive about some anti-alcohol effects of lithium. Favorable anecdotal reports have continued to appear, and there is every indication that lithium may be prescribed quite widely in alcoholism. It is remarkable that since lithium has a very specific therapeutic effect on mania and a much less clear effect on depression that no one has previously reported on manic (or Bipolar) patients with problem drinking or alcoholism in a systematic fashion. This lack of data may perhaps result from difficulty in locating such a population. In a previous publication, two of the present authors reported little benefit on alcohol symptoms in a small number of patients with mania who they treated for 6 or more months. In the present study, we have extended this first report to a group of 20 Bipolar or manic patients with concurrent diagnosis of alcoholism or abusive drinking, all of whom were followed on lithium for not less than one year.

Acute effects of disulfiram on anxiety levels of chronic alcoholics.

30 male alcoholics, who were anxious by clinical research standards, received either disulfiram (250 mg) or placebo daily for 1 week. There are claims that disulfiram has a sedative effect and that this has some useful antianxiety action, but this had not been tested in a controlled manner. There were no significant differences in the mean changes between the disulfiram and placebo groups as determined by any of the tests used. Both the Zung self-rating anxiety scale and the anxiety scale of the profile of mood states indicated ( p less than 0.05) that those receiving disulfiram were extreme reactors as compared to the placebo group; those receiving disulfiram either became more anxious or experienced the most improvement. The study suggests that disulfiram may selectively relieve or increase the anxiety level of subgroups of subjects, but does not delineate a predictor response.

Are all recently detoxified alcoholics depressed?

Reports of the prevalence of depression among alcoholics vary from 3% to 98%; the authors attribute this variation to the use of different diagnostic criteria. They used clinical diagnosis, the Hamilton Depression Rating Scale, the Zung Self-Rating Depression Scale, and the MMPI to diagnose 35 men recently withdrawn from alcohol. The percentages of depression diagnosed were 8.6%, 28%, 66%, and 43%, respectively. The authors point out that the Hamilton, Zung, and MMPI scales are not diagnostic of depression in themselves and that accurate diagnosis of depression in alcoholics will reduce inappropriate treatment of nondepressed alcoholics and ensure careful treatment of the truly depressed.

Selection among benzodiazepines for alcohol withdrawal.

Four pharmacologic differences among the benzodiazepines, which are the drugs of choice to conduct alcohol withdrawal, guide selection of the appropriate one: chlordiazepoxide, diazepam, oxazepam, or chlorazepate. Bases for selection include: (1) availability of other than oral dosage forms; (2) differences in additive effect with alcohol in producing central nervous system depression; (3) differences in anticonvulsant effect; and (4) differences in duration of effect in the body (ie, half-life). Decreasing dosage schedules are preferred to a steady dosage. Illustrative dosage schedules for using chlordiazepoxide and diazepam to conduct alcohol withdrawal are given.