Regulatory action of prolactin on the in vitro growth of CD34+ve human hemopoietic progenitor cells

The pituitary hormone prolactin (Prl) is known to act as a local regulator of immune cell function, and Prl-binding receptors (Prl-R) have been described to share distinctive features with the members of the newly described cytokine/hemopoietin receptor superfamily. Here we show that the hormone can functionally interact with lineage-specific hemopoietic factors. When highly purified progenitor cells (CD34+ve) were seeded in semisolid methylcellulose cultures in the presence of interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), and erythropoietin (Epo), a selective enhancing effect of Prl on the formation of colony forming unit-granulocyte (CFU-G) and burst forming unit-erythroid (BFU-E) colonies was observed. The effect of the hormone was plotted as a bell shaped curve, with the optimal response at the supraphysiological concentration of 50 ng/ml. Limiting dilution analysis showed that Prl acted directly on hemopoietic progenitors. This was confirmed by the observation on the CD34+ve cells of Prl-binding sites reacting with the specific monoclonal antibodies (mAbs), U5 and PrR-7A. Immunoprecipitation of the metabolically labeled CD34+ve cells with the PrR-7A mAb revealed a structure of 43 kD under reducing conditions. Analysis of the early events associated with the Prl/Prl-R interaction showed an increased number of cells engaged in DNA and hemoglobin synthesis. Enhanced erythroid differentiation of CD34+ve cells in the presence of Prl was secondary to upmodulation of receptors for the lineage-specific factor Epo. Together these data demonstrate the existence of a functional interplay between Prl. and hemopoietic factors. © 1995 Wiley-Liss, Inc.     

A common β hexosaminidase gene mutation in adult Sandhoff disease patients

-Hexosaminidase gene mutations were analyzed in two adult-onset Sandhoff disease Italian patients by PCR analysis of a common known mutation (5) and by heteroduplex analysis of genomic and RT-PCR DNA fragments, covering the whole gene. The patients' genotypes were 5/C1214T, and G890A/C1214T, respectively. As mutation C1214T (Pro405Leu) is also present in the other two late-onset cases so far described, we suggest that C1214T is a common mutation in this type of Sandhoff disease. Mutation G890A (Cys297Tyr) is a novel mutation which presumably causes altered processing of the pro chain.     

A new biallelic DNA polymorphism of the human COL5A1 gene

A cDNA probe of the human COL5A1 gene detects a frequent biallelic PstI polymorphism. Allele A has a frequency of 54% whereas that of allele B is 46%. This restriction fragment length polymorphism provides a useful marker for linkage analysis in 9q34.3.     

Ferredoxin—ferredoxin NADP reductase interaction: Catalytic differences between the soluble and thylakoid-bound complex

Ferredoxin-NADP reductase (FNR) and ferredoxin form a complex when the former is membrane-bound as they do when both components are in solution, with the same dissociation constant. The rate constant of NADP photoreduction, first order with respect to the complex, is more than 20-times higher when FNR is membrane-bound than when the enzyme is in solution. The Arrhenius activation energy is identical in both conditions. These observations are interpreted in terms of ‘entropic catalysis’ of NADP reduction by the thylakoid-bound FNR.     

Regulatory properties of rabbit red blood cell hexokinase at conditions close to physiological

The true level of hexokinase in rabbit erythrocytes was determined by three different methods, including the spectrophotometric glucose-6-phosphate dehydrogenase coupled assay and a new radioisotopic assay. The value found at 37°C (pH 7.2) was 10.23±1.90 μmol/h per ml red blood cells, which is lower than previously reported values. More than 40 cellular components of the rabbit erythrocytes were tested for their effects on the enzyme. Their intracellular concentrations were also determined. Several of these compounds were found to be competitive inhibitors of the enzyme with respect to Mg·ATP^2?. Furthermore, reduced glutathione at a concentration of 1 mM was able to maintain hexokinase in the reduced state with full catalytic activity. The ability of orthophosphate to remove the inhibition of some phosphorylated compounds was examined under conditions similar to cellular (pH 7.2 and 50 μM of orthophosphate) and found to be of no practical interest. In contrast, the binding of ATP^4? and 2,3-diphosphoglycerate to the rabbit hemoglobin significantly modifies their intracellular concentrations and the formation of the respective Mg complexes. The pH-dependence of the reaction velocity and of the kinetic properties of the enzyme in different buffer systems were also considered. This information was computerized, and the rate of glucose phosphorylation in the presence of the mentioned compounds was determined. The value obtained, 1.94±0.02 μmol/h per ml red blood cells, is practically identical to the measured rate of glucose utilization by intact rabbit erythrocytes (1.92±0.3 μmol/h per ml red blood cells). These results provide further evidence for the central role of hexokinase in the regulation of red blood cell glycolysis.     

Rabbit red blood cell hexokinase

Summary Rabbit hexokinase (EC 2.7.1.1) has been shown to exist in reticulocytes as two distinct molecular forms, designated hexokinase Ia and Ib, but only one of these was consistently present in mature red cells. In vivo, hexokinase la and Ib show a decay rate of 3 and 8% a day, respectively, while in vitro they show a similar stability.The possibility that the proteolytic activities of the reticulocyte could be responsible for the fast decay of hexokinase was investigated. No differences were found in the decay rates of hexokinase la and Ib during in vitro reticulocyte maturation in presence or absence of proteolytic inhibitors. Contrariwise, many findings indicate the ATP-dependent proteolytic system of the reticulocyte as a possible mechanism. In fact, the decay of hexokinase and the degradation of 3H-globins are both stimulated by ATP and ubiquitin; they show similar kinetic properties and both disappear during reticulocyte maturation.The cellular localization of hexokinase la and Ib was shown to be responsible for the differences found between their decay rates.Abbreviations PMSF phenylmethylsulfonyl fluoride - TPCK 1-1-tosylamide-2-phenylethyl-chloromethyl ketone - TLCK N -p-tosyl-L-lysine chloromethyl ketone     

pK determinations via pH-mobility curves obtained by isoelectric focusing-electrophoresis: Theory and experimental verification

Basic equations have been derived linking the electrophoretic migration in a stationary pH gradient of simple, singly charged cations or anions and of mono- mono- valent ampholytes with the pKs of their ionizable groups. In the case of diprotic ampholytes, an equation and a curve are described calculating a correction factor to be applied to the mobility measurements, accounting for the influence of the opposite charge species on the mobility curve of the ion being measured. This correction factor is a function of ΔpK and increases exponentially with decreasing values of ΔpK. These theoretical considerations have been experimentally verified by running pH-mobility curves of colored compounds, such as methyl red, neutral red and dexorubicin. The pKs thus measured were in excellent agreement with the pKs obtained independently by spectrophotometric titrations.