Preparation of lipid emulsions by pressure extrusion

Lipid emulsions consisting of a surface monolayer of phospholipid enclosing a core of neutral lipids have been prepared by repeated extrusion through polycarbonate filters of defined pore size. Particle size, as measured by photon correlation spectroscopy, decreases on successive passes through a 100 nm filter, reaching a near constant value (130-150 nm) after 4 passes. A corresponding decrease in the standard deviation of the particle size distribution occurs during this process. The recovery of lipids, especially of cholesterol and cholesterol ester, is improved if the emulsion is sonicated before extrusion through filters. [31P]-NMR and fluorescence techniques are used to confirm that the resulting structures are emulsions rather than lipid bilayers.     

Selective effect of zinc compared to other divalent metals on L-triiodothyronine binding to rat c-erbA alpha and beta proteins

The effects of zinc and other divalent metals on the [125I]T3 binding to rat c-erbA alpha and beta recombinant proteins were assessed. The addition of ZnCl2 caused a reversible and dose-dependent inhibition of [125I]T3 binding to rc-erbA beta proteins with half maximum inhibition occurring at 50-100 microM, but no significant effect on [125I]T3 binding to rc-erbA alpha under the same assay conditions. Scatchard analysis revealed a decrease in [125I]T3 binding capacity to beta protein without marked change in Kd values in presence of zinc. Moreover, significant inhibitions of [125I]T3 binding to both alpha and beta proteins were observed in the presence of 100 microM of either MnCl2, CdCl2 or CuCl2, but not MgCl2. Thus, the selective effect of zinc compared to other divalent metals to inhibit T3 binding to rc-erbA beta, but not alpha, proteins was documented and suggest a possible regulatory role for zinc in modulating the intracellular action of thyroid hormone.     

Papillary carcinoma of thyroid: classical and variants

Papillary carcinoma, the commonest primary cancer of thyroid, exhibits a broad morphological spectrum. In this review, the clinicopathological features of papillary carcinoma, classical and its variants (follicular, solid, cribriform, variant with exuberant nodular fasciitis-like stroma, encapsulated, diffuse sclerosing, diffuse follicular, tall cell, columnar cell, oxyphil cell, "dedifferentiated", occult, latent and microcarcinoma) are summarized.     

Eight small subunits of Euglena ribulose 1-5 bisphosphate carboxylase/oxygenase are translated from a large mRNA as a polyprotein.

The small subunit (SSU) of ribulose 1-5 bisphosphate carboxylase/oxygenase is a 15 kd protein in Euglena gracilis. The protein is synthesized as a 130 kd precursor as shown by immunoprecipitation of in vitro translation products and confirmed by immunoprecipitation of in vivo pulse-labeled Euglena proteins. From the published SSU amino acid sequence, an oligonucleotide was synthesized that specifically hybridizes to a large mRNA whose length (approximately 4.3 kb) is consistent with the precursor size. The complete nucleotide sequence of the SSU mRNA was obtained by sequencing a cDNA clone from a lambda gt11 library and completed by direct mRNA sequencing. We report for the first time the complete sequence of a large mRNA and show that it encodes eight consecutive SSU mature molecules. The deduced precursor amino acid sequence shows that the amino terminus of the first SSU molecule is preceded by a 134 amino acid peptide which is cleaved during the maturation process. This long transit peptide exhibits features characteristic of signal peptides involved in the secretion of proteins through the endoplasmic reticulum. This is in agreement with the idea that the third (outer) membrane of the Euglena chloroplast envelope is of endoplasmic reticulum origin.     

Distinct tyrosine phosphorylation sites in ZAP-70 mediate activation and negative regulation of antigen receptor function.

Biochemical and genetic evidence has implicated two families of protein tyrosine kinases (PTKs), the Src- and Syk-PTKs, in T- and B-cell antigen receptor signaling. ZAP-70 is a member of the Syk-PTKs that associates with the T-cell antigen receptor and undergoes tyrosine phosphorylation following receptor activation. Three tyrosine residues, Tyr-292, -492, and -493, have been identified as sites of phosphorylation following T-cell antigen receptor engagement. Utilizing ZAP-70- and Syk-deficient lymphocytes (Syk-DT40 cells), we provide biochemical and functional evidence that heterologous trans-phosphorylation of Tyr-493 by a Src-PTK is required for antigen receptor-mediated activation of both the calcium and ras pathways. In contrast, cells expressing mutations at Tyr-292 or -492 demonstrate hyperactive T- and B-cell antigen receptor phenotypes. Thus, phosphorylation of ZAP-70 mediates both activation and inactivation of antigen receptor signaling.     

Modulation of Calmodulin mRNA and Protein Levels in Barley Aleurone

Changes in calmodulin (CaM) mRNA and protein were investigated in aleurone layers of barley (Hordeum vulgare L. cv Himalaya) incubated in the presence and absence of calcium, gibberellic acid (GA3), and abscisic acid (ABA). CaM mRNA levels increased rapidly and transiently following incubation of aleurone layers in H2O, CaCl2, or GA3. The increase in CaM mRNA was prevented by ABA. This increase in CaM mRNA was brought about by physical stimulation during removal of the starchy endosperm from the aleurone layer. CaM protein levels did not increase in response to physical stimulation. Only incubation in GA3 plus CaCl2 brought about a rapid increase in CaM protein levels in the aleurone cell. ABA reduced the level of CaM protein below that found at the beginning of the incubation period. The rise in CaM protein preceded increases in the synthesis and secretion of [alpha]-amylase. Immunocytochemistry with monoclonal antibodies to carrot and mung bean CaM was used to localize CaM in aleurone protoplasts. Monoclonal antibodies to tubulin and polyclonal antibodies to tonoplast intrinsic protein and malate synthase were used as controls. CaM was localized to the nucleus, the vacuolar membrane, and the cytosol, but was not associated with microtubules.     

The LIM domain-containing Dbm1 GTPase-activating protein is required for normal cellular morphogenesis in Saccharomyces cerevisiae.

Normal cell growth in the yeast Saccharomyces cerevisiae involves the selection of genetically determined bud sites where most growth is localized. Previous studies have shown that BEM2, which encodes a GTPase-activating protein (GAP) that is specific for the Rho-type GTPase Rho1p in vitro, is required for proper bud site selection and bud emergence. We show here that DBM1, which encodes another putative Rho-type GAP with two tandemly arranged cysteine-rich LIM domains, also is needed for proper bud site selection, as haploid cells lacking Dbm1p bud predominantly in a bipolar, rather than the normal axial, manner. Furthermore, yeast cells lacking both Bem2p and Dbm1p are inviable. The nonaxial budding defect of dbm1 mutants can be rescued partially by overproduction of Bem3p and is exacerbated by its absence. Since Bem3p has previously been shown to function as a GAP for Cdc42p, and also less efficiently for Rho1p, our results suggest that Dbm1p, like Bem2p and Bem3p, may function in vivo as a GAP for Cdc42p and/or Rho1p. Both LIM domains of Dbm1p are essential for its normal function. Point mutations that alter single conserved cysteine residues within either LIM domain result in mutant forms of Dbm1p that can no longer function in bud site selection but instead are capable of rescuing the inviability of bem2 mutants at 35 degrees C.     

Cloning and expression of mouse 60 kDa ribonucleoprotein SS-A/Ro

Autoantibodies to SS-A/Ro are among the most common found in sera of patients with systemic rheumatic diseases. These autoimmune diseases can affect various organ systems of the body and are variable in their manifestations and presentation. One of the autoimmune targets is the 60 kDa SS-A/Ro protein known to be associated with small cytoplasmic Y RNAs. To study systematically the expression of the protein, we have cloned the mouse full length 60 kDa SS-A/Ro cDNA using 5′ RACE based on a cDNA sequence reported in the mouse genome project. The recombinant protein derived from the putative full-length construct was shown to react with human prototype anti-SS-A/Ro serum Ge in western blot and immunoprecipitation and comigrated with cellular 60 kDa SS-A/Ro protein in 3T3 cells. Cellular expression, measured by RT-PCR, was highest in mouse brain, followed by lung, muscle, kindney and heart. Lower levels were found in testis, liver and spleen. Like the human 60 kDa SS-A/Ro protein, the deduced mouse homolog has 538 amino acids. Sequence analysis showed 89.9% identity and 95.0% similarity between the mouse and human proteins.     

Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures

Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10⁹ cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.     

Identification of Biochemical Defects in Pancreatic Islets of fa/fa Rats: A Developmental Study

KIBENGE, MOLLY T AND CATHERINE B CHAN. Identification of biochemical defects in pancreatic islets of fa/fa rats: a developmental study. Obes Res. 1995;3:171–178. Adult obese (fa/fa) Zucker rats hypersecrete insulin in response to glucose and other secretagogues. Functional changes in islet ot2-adrenoceptors (8) and glycolytic regulation (9) have been reported. In this study, the development of these biochemical lesions in islets isolated from suckling (3 week old) and weanling (5 week old) lean and fa/fa rats was investigated and compared to results in adult animals. Glucose (15 mM)-induced insulin secretion was inhibited by mannoheptulose (MH) in lean (n=8) but not fa/fa (n=10) adult rats, indicating loss of sensitivity of glucokinase to competitive inhibition. Sensitivity to MH was somewhat reduced in the islets of 3- and 5-week-old fa/fa (n=7 and 12) compared to lean (n=15 and 9) rats, requiring 30–100 fold higher concentrations to achieve significant inhibition. At 3 weeks of age fa/fa rats did not differ from lean controls in either islet insulin content or body weight, but both parameters were increased in fa/fa rats by 5 weeks. The presence of altered α₂-adrenoceptor function in fa/fa rats could not be confirmed in this study. Unlike the previous report, prazosin did not antagonize α₂-agonist mediated inhibition of insulin secretion. The presence of defective regulation of the glycolytic pathway by mannoheptulose in suckling and weanling rats may contribute to development of hyperinsulinemia in fa/fa rats.