A Technique for Simultaneous Antikinetochore Immunofluorescence Staining and Q-Banding in Chromosomes from Human Lymphocytes

Antikinetochore immunofluorescence staining has been used in several studies to determine whether a second kinetochore is present, active, or both, in multicentric chromosomes. All of these studies have used tissue culture cells, and contended with the problem of obtaining well spread, banded metaphase chromosomes without affecting the kinetochore staining. We have adapted hypotonic, centrifugation and chromosome staining techniqnes to obtain simultaneous Q-banding and bright kinetochore staining of chromosomes from human lymphocytes.     

Time and money in pediatric practice

The types of services provided by pediatricians in Ontario and the payment they received from OHIP for a three-month period in 1972 are examined. Pediatricians provided 4.8% of the total of 16.3 million services and received 3.3% of the total $117.7 million payment. Most of the services were provided in ambulatory settings. Skin testing for allergy and hyposensitization accounted for 42% of the total of pediatric services. More than two thirds of pediatricians were engaged in provision of services which could be classified as primary care. Indeed, most of the services provided were of a primary care nature.There is a pressing need for redifining the pediatrician''s role in our health-care delivery system and for a new look at his education.     

Influence of microbial concentration on the rheology of non-Newtonian fermentation broths

The objective of this study was to quantify the effect of fungal biomass concentration on the rheology of non-Newtonian fermentation systems. Batch fermentations of Penicillium chrysogenum were carried out with glucose as the sole carbon source. The flow behavior of the system was characterized at various fermentation times and was adequately described by the power-law model. The apparent viscosity of the fermentation broth was significantly affected by biomass concentrations in the fermenter. Fermentation broths containing 17.71 g/l biomass as dry weight were characterized by an apparent viscosity of 0.25 Pa s at a shear rate of 50 s⁻¹. Microbial concentration also affected the power-law flow-behavior index and the consistency index. The value of the consistency index ranged from 0.002 Pa s ⁿ at a biomass concentration of 0.1 g/l to 6.14 Pa s ⁿ at a biomass concentration of 17.71 g/l. The flow-behavior index decreased from an initial value of 1 to a final value of 0.17. Simple empirical correlations have been proposed to quantify the dependence of the power-law parameters on fungal biomass concentration. Experimental data obtained in this study were accurately described by these correlations. The general applicability of these relationships was tested, using previously published rheological data on Aspergillus awamori and Aspergillus niger fermentation broths, and good agreement was seen between experimental data and the predictions from the empirical correlations. Received: 24 March 1998 / Received revision: 10 September 1998 / Accepted: 16 October 1998     

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery.

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 microM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) (100 microM) or inhibiting PDI with bacitracin (5mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 microM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 microM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.     

Calmodulin interacts with the V2 vasopressin receptor: elimination of binding to the C terminus also eliminates arginine vasopressin-stimulated elevation of intracellular calcium

To identify molecules that might contribute to V2 vasopressin receptor (V2R) trafficking or signaling, we searched for novel interacting proteins with this receptor. Preliminary data, using the V2R C terminus as bait in a yeast two-hybrid screen, revealed calmodulin as a binding partner. Because calmodulin interacts with other G protein-coupled receptors, we explored this interaction and its possible functional relevance in greater detail. A Ca2+ -dependent interaction occurs between calmodulin-linked agarose and the holo-V2R as well as the V2R C terminus. Truncation and site-directed mutagenesis of the V2R C terminus revealed an involvement of an RGR sequence in this interaction. NMR studies showed that a peptide fragment of the V2R C terminus containing the RGR sequence binds to calmodulin in a Ca2+ -dependent manner with a Kd < or =1.5 microm; concentration-dependent binding of the V2R C terminus to calmodulin-agarose was used to estimate a Kd value of approximately 200 nm for this entire C-terminal sequence as expressed in mammalian cells. Madin-Darby canine kidney II cells stably expressing either wild type or a mutant V2R, in which the RGR C-terminal sequence was mutated to alanines (AAA V2R), revealed that the steady-state localization and agonist-induced internalization of the AAA V2R resembled that of the wild type V2R in polarized Madin-Darby canine kidney II cells. V2R binding of agonist similarly was unchanged in the AAA V2R, as was the concentration response for arginine vasopressin (AVP)-stimulated cAMP accumulation. Most interestingly, AVP-induced increases in intracellular Ca2+ observed for the wild type V2R were virtually eliminated for the AAA V2R. Taken together, the data suggest that a C-terminal region of the V2R important for calmodulin interaction is also important in modulation of V2R elevation of intracellular Ca2+, a prerequisite for AVP-induced fusion of aquaporin-containing vesicles with the apical surface of renal epithelial cells.     

Critical role of T-loop and H-motif phosphorylation in the regulation of S6 kinase 1 by the tuberous sclerosis complex

The tuberous sclerosis gene products Tsc1 and Tsc2 behave as tumor suppressors by restricting cell growth, a function conserved among metazoans. Recent evidence has indicated that hyperactivation of S6 kinase 1 (S6K1) may represent an important biochemical change in the development of tuberous sclerosis-associated lesions. We show here that deletion of either Tsc1 or Tsc2 or expression of the Rheb (Ras homolog enriched in brain) GTPase leads to hyperphosphorylation of S6K1 at a subset of regulatory sites, particularly those of two essential residues functionally conserved among AGC superfamily serine/threonine kinases, i.e. the activation loop (T-loop; Thr-229) and the hydrophobic motif (H-motif; Thr-389). These sites are reciprocally and dose-dependently regulated when S6K1 is coexpressed with the Tsc1-Tsc2 complex. Mutations that render S6K1 mTOR (mammalian target of rapamycin)-resistant also protect S6K1 activity and phosphorylation from down-regulation by Tsc1/2. We demonstrate that two disease-associated mutations in Tsc2 fail to negatively regulate S6K1 activity concomitant with a failure to modify T-loop and H-motif phosphorylation. Finally, we identify one pathological Tsc2 mutation that retains its ability to negatively regulate S6K1, suggesting that, in some cases, tuberous sclerosis may develop independently of S6K1 hyperactivation. These results also highlight the importance of dual control of T-loop and H-motif phosphorylation of S6K1 by the Tsc1-Tsc2 complex.