Combined use of nonmyelosuppressive nitrosourea analogues with hydroxyurea in the induction of F-cell production in a human erythroleukemic cell line.

Objective: Although hydroxyurea (HU) has been used clinically to treat patients with sickle cell disease (SCD), not all patients benefit from HU treatment due to its toxicity. The objective of this study was to investigate the effectiveness of the use of two new Hb F-inducing nitrosourea analogues, 2-[3-(2-methyl, 2-nitroso) ureido]-2-deoxy-D-glucopyranose (MNGU) and 2-[3-(2-chloroethyl) ureido]-2-deoxy-D-glucopyranose (CGU), in combination with HU in K562 cells or erythroid progenitors.

Materials And Methods: After K562 cells were cultured with different concentrations of HU with CGU or MNGU, aliquots of the cells were obtained to determine the total (benzidine-positive) hemoglobin level, number of F cells, and Hb F level.

Intracellular localization of the 90 kDA heat shock protein (HSP90alpha) determined by expression of a EGFP-HSP90alpha-fusion protein in unstressed and heat stressed 3T3 cells.

Heat shock protein 90 (Hsp90) is an abundant protein and essential for all eukaryotic cells. The expression of Hsp90 is further enhanced after exposure to stress factors, e.g.

Evidence that the novobiocin-sensitive ATP-binding site of the heat shock protein 90 (hsp90) is necessary for its autophosphorylation.

The 90kDa heat shock protein (Hsp90) is one of the most abundant protein and essential for all eukaryotic cells. Many proteins require the interaction with Hsp90 for proper function. Upon heat stress the expression level of Hsp90 is even enhanced.

A novel function for the 90 kDa heat-shock protein (Hsp90): facilitating nuclear export of 60 S ribosomal subunits.

Ribosomal subunits are assembled in the nucleus, and mature 40 S and 60 S subunits are exported stoichiometrically into the cytoplasm. The nuclear export of ribosomal subunits is a unidirectional, saturable and energy-dependent process. An in vitro assay for the nuclear export of 60 S ribosomal subunits involves the use of resealed nuclear envelopes.

Isolation and quantification of the heat shock protein 90 alpha and beta isoforms from rat liver.

Heat shock protein 90 (Hsp90) is an abundant cytosolic protein. In higher eukaryotes two isoforms of Hsp90 exist, Hsp90 alpha and Hsp90 beta. Hsp90 was purified from rat liver and after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a double band at about 90 kDa.

Hydroxyurea-induced oxidative damage of normal and sickle cell hemoglobins in vitro: amelioration by radical scavengers.

Hydroxyurea (HU) induces fetal hemoglobin (Hb F) production in patients with sickle cell anemia. The therapeutic dosage of HU used for Hb F induction often elicits myelosuppression, which becomes its major associated complication. We examined the effect of HU on hemoglobin modulation and the role of radical scavengers on these induced changes.

Enhancement of hemoglobin and F-cell production by targeting growth inhibition and differentiation of K562 cells with ribonucleotide reductase inhibitors (didox and trimidox) in combination with streptozotocin.

Upon appropriate drug treatment, the human erythroleukemic K562 cells have been shown to produce hemoglobin and F-cells. Fetal hemoglobin (Hb F) inhibits the polymerization events of sickle hemoglobin (Hb S), thereby ameliorating the clinical symptoms of sickle cell disease. Ribonucleotide reductase inhibitors (RRIs) have been shown to inhibit the growth of myeloid leukemia cells leading to the production of Hb F upon differentiation.

Trimidox-mediated morphological changes during erythroid differentiation is associated with the stimulation of hemoglobin and F-cell production in human K562 cells.

Trimidox (3,4,5-trihdroxybenzamidoxime) has been shown to reduce the activity of ribonucleotide reductase with accompanied growth inhibition and differentiation of mammalian cells. Hydroxyurea (HU) is the only ribonucleotide reductase inhibitor in clinical use for the treatment and management of sickle cell anemia, since this compound increases fetal hemoglobin (Hb F) production: a potent inhibitor of sickle hemoglobin (Hb SS) polymerization. However, the main limitations of HU is its lack of potency, myelosuppression and short half life.

Three different actions of phenylglyoxal on band 3 protein-mediated anion transport across the red blood cell membrane.

Phenylglyoxalation of the red blood cell membrane leads to three superimposed effects on band 3 protein-mediated anion equilibrium exchange as measured by means of radiosulfate: (1) a shift of the curve relating transport activity to pH towards lower pH values, possibly in combination with an increase of the maximal transport activity. This is accompanied by effect (2), the abolishment of a chloride-stimulated component of anion transport seen at low pH values. Effect (3) consists of inhibition of anion equilibrium exchange.

Export of ribosomal subunits from resealed rat liver nuclear envelopes.

We have previously described the rat liver resealed nuclear envelope model system for the study of the selective import of nuclear proteins, and the export of poly(A)-containing mRNA [Riedel, N., Bachmann, M., Richter, H.

Cross-linking of nucleic acids to proteins. Modified poly(A) as mRNA for Escherichia coli ribosomes.

Poly(adenylic acid) was modified by methylchlorotetrolic ester in a reproducible and defined content of the derivatized bases. The nucleic acid derivative is protein reactive and was coupled to 70S ribosomes from Escherichia coli, in order to identify proteins along the mRNA pathway. The binding of the label becomes specific under the direction of tRNA(Lys) and is then almost exclusively located on the small subunit.

4'-Amino-benzamido-taurocholic acid selectively solubilizes glycosyl-phosphatidylinositol-anchored membrane proteins and improves lipolytic cleavage of their membrane anchors by specific phospholipases.

Glycosyl-phosphatidylinositol-anchored membrane proteins (GPI-proteins) are normally identified either by cleavage of the lipid anchor using (glycosyl)phosphatidylinositol-specific phospholipases C or D (GPI-PLs) or by metabolic labeling of the lipid moiety with specific building blocks. Therefore, methods for discrimination between transmembrane proteins and GPI-proteins on the basis of their physicochemical properties are desirable. Here we are presenting a selective extraction method for typical well-characterized mammalian GPI-proteins, e.

Purification and partial sequence of proteins involved in the cholic acid transport into rat liver hepatocytes.

Two proteins, in previous work labeled by affinity markers derived from taurocholic acid, were purified and partially sequenced. Antibodies were raised against purified proteins, and cross-reactions were carefully checked. The influence of these antibodies upon taurocholic acid import into vesicles from rat liver plasma membranes was measured, and showed a distinct inhibition of transport in the case of the 54 kD protein.

[Age-dependent changes in mRNA transport (nucleus-cytoplasm)].

Transport of mRNA from nucleus to cytoplasm is an ATP-dependent process which occurs strictly vectorially. Because the mRNA is structurally bound during transport, mRNA transport is a "solid-state" process consisting of i) mRNA release from the nuclear matrix, ii) mRNA translocation through the nuclear pore, and iii) cytoskeletal binding. We identified and purified the following components involved in the translocation step: i) the nuclear envelope (NE) nucleoside triphosphatase (NTPase) which is stimulated by the 3'poly(A) tail of mRNA, ii) the poly(A)-recognizing mRNA carrier, iii) the NE protein kinase, and iv) the NE phosphatase.

Characterization and chemical modification of the Na(+)-dependent bile-acid transport system in brush-border membrane vesicles from rabbit ileum.

The Na(+)-dependent uptake system for bile acids in the ileum from rabbit small intestine was characterized using brush-border membrane vesicles. The uptake of [3H]taurocholate into vesicles prepared from the terminal ileum showed an overshoot uptake in the presence of an inwardly-directed Na(+)-gradient ([Na+]out > [Na+]in), in contrast to vesicles prepared from the jejunum. The Na(+)-dependent [3H]taurocholate uptake was cis-inhibited by natural bile acid derivatives, whereas cholephilic organic compounds, such as phalloidin, bromosulphophthalein, bilirubin, indocyanine green or DIDS - all interfering with hepatic bile-acid uptake - did not show a significant inhibitory effect.

Covalent attachment of ribonucleic acids to proteins.

As a prerequisite for the synthesis of affinity labels, we describe methods to couple histones to ribonucleic acids. For the synthesis of these covalent hybrid molecules, we used a population of histones H1, H2A, H2B, H3, and H4 from calf thymus and polyadenylic acid with an average chain length of up to 260-280 bases, representing the size of poly(A)-tails from mature mRNAs. Three methods were investigated.

Interleukin-2 and blood brain barrier in cats: pharmacokinetics and tolerance following intrathecal and intravenous administration.

Single bolus doses of glycosylated human interleukin-2 (n IL-2) in the range of 2.8 x 10(3) to 2.0 x 10(6) IU/kg were administered to anesthesized cats via the cephalic vein (n = 10) or using suboccipital puncture (n = 8).

A strongly basic protein of the MAP2 family copolymerizes with tubulin and induces polymerization.

The family of microtubuli-associated proteins of approximately 300 kD molecular weight (MAP2) from porcine brain was fractionated into components of neutral isoelectric point and one polypeptide of strongly basic nature. Both fractions are able to induce the polymerization of purified porcine brain tubulin. In the case of the fractions of an isoelectric point of 7.

Poly(A) binding proteins located at the inner surface of resealed nuclear envelopes.

We have used a photoreactive cross-linking reagent, poly(A/8-N3-A) (a poly(A) of average molecular mass of 100 kDa in which 5-10% of the A residues are replaced by 8-N3-A), to label poly(A) binding proteins of rat liver nuclear envelopes. This reagent was prepared by polymerizing a mixture of ADP and 8-N3-ADP with polynucleotide phosphorylase. The purified poly(A) was labeled in the 5'-position with a 32P group.

Interaction of a nuclear location signal with isolated nuclear envelopes and identification of signal-binding proteins by photoaffinity labeling.

The nuclear envelope (NE) separates the two major compartments of eukaryotic cells, the nucleus and the cytoplasm. Recent studies suggest that the uptake of nuclear proteins into the nucleus is initiated by binding of nuclear location signals (NLSs) contained within these proteins to receptors in the NE, followed by translocation through the nuclear pore complex. To examine the binding step without interference from intranuclear events, we have used a system consisting of (i) purified rat liver NEs fixed onto glass slides and (ii) the prototype simian virus 40 large T antigen (SV40 T) NLS conjugated to nonnuclear carrier proteins, and we have visualized the receptor-ligand interaction by indirect immunofluorescence.

Major proteolytic fragments of the murine band 3 protein as obtained after in situ proteolysis.

Proteolytic fragments of murine band 3 were produced by exposure to extracellular chymotrypsin and intracellular trypsin. The ensuing proteolytic fragments were isolated, their N-terminal sequences were determined and their locations in the known amino acid sequence of murine band 3 established. Equivalents of the human 60, 35 and 17 kDa fragments were obtained through the cleavage sites were situated at locations that are not strictly homologous to the corresponding cleavage sites in human band 3, although all of them were near such sites.

Bile acid binding proteins in hepatocellular membranes of newborn and adult rats. Identification of transport proteins with azidobenzamidotauro[14C]cholate ([14C]ABATC).

Neonatal hepatocytes are less active in uptake of bile acids than are mature hepatocytes. This phenomenon has been further investigated by transport studies with azidobenzamidotaurocholate (ABATC). Taurocholate, cholate and the photolabile ABATC were taken up by liver cells of adult rats by a sodium-dependent and by an additional sodium-independent mechanism.

The anion-transport inhibitor H2DIDS cross-links hemoglobin interdimerically and enhances oxygen unloading.

Human hemoglobin treated with equal concentrations of the anion-transport inhibitor H2DIDS produces a right shift in the oxygen dissociation curve. concomitantly, the Hill coefficient is reduced from n = 2.7 to 2.

Artificial dimers of native actin: preparation and properties in biological functions.

With the aid of tartryl-bis-epsilon-aminocaprylazide artificial dimers were produced from F actin from rabbit striated muscle. These derivatives will not polymerize by themselves but are able to copolymerize fully with native G actin. By modification of a single side chain per dimer, this copolymerization was completely inhibited.