Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells.

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.     

Effect of DTNB on glutamate dehydrogenase activity in root and shoot extracts of maize seedlings

NADH specific glutamate dehydrogenase (GDH) activity was examined in roots and shoots of maize seedlings grown in half-strength Hoagland’s solution containing NH₄NO₃ as sole nitrogen source under irradiance of 60 W m⁻² and temperature of 25±2°C. When 5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB) was supplied to the assay mixture, it inhibited NADH-GDH activity in both roots and shoots, irrespective of whether the enzymes were extracted from light- or dark-treated roots and shoots. In each case the inhibition increased with the increase in DTNB concentration. At the maximum concentration of DTNB used (20 μM) the inhibition of shoot NADH-GDH was more pronounced than inhibition of root enzyme. This indicated differences in shoot and root NADH-GDH.     

Effect of hypocholesterolemic agents of plant origin on catecholamine biosynthesis in normal and cholesterol fed rabbits

The effect of lipid lowering agents of plant origin garlic oil and guggulipid on the levels of catecholamine and dopamine Β-hydroxylase activity of normal and cholesterol fed rabbit tissues has been studied. The catecholamine levels and enzyme activity were found to be decreased in cholesterol (500 mg/kg body wt) fed animals. The feeding of garlic oil (5 mg/kg body wt) and guggulipid (100 mg/kg body wt) an exudate ofCommiphora mukul, to normal rabbits caused significant increase in the dopamine-Β-hydroxylase activity and catecholamine levels, while the feed helped the hypercholesterolemic rabbits to recover the decrease in catecholamine biosynthesis C.D.R.I. Communication No. 3435.     

Electron microscopic studies on liver 5-aminolaevulinate synthase

The structure of chick embryo liver 5-aminolaevulinate synthase has been examined by electron microscopic studies using negative staining. From the different projections of the enzyme particles observed in electron micrographs, a model for the enzyme molecule has been proposed. In this model, an enzyme molecule consists of two curved and identical subunits associated in opposite polarities. From the dimensions of an enzyme molecule subunit measured from electron micrographs, the relative molecular mass of each subunit is estimated to be 70 000.     

Affinity chromatography of terminal deoxynucleotidyl tranferase from calf thymus and human leukemic cells on a solid-phase immunoadsorbent

A solid-phase immunoadsorbent specific for terminal deoxynucleotidyl transferase has been prepared. The enzyme from calf thymus and acute lymphoblastic leukemia cells binds to columns of this material. Bound enzyme can be eluted in an active form. Selective and rapid purification of terminal deoxynucleotidyl transferase from crude extracts of cells containing this enzyme can be achieved by this method since the immunoadsorbent has no affinity for other cellular DNA polymerases.     

Measurement of nitrite reductase in leaf tissue of Vigna mungo

The enzyme nitrite reductase (EC 1.6.6.4) is generally assayed in terms of disappearance of nitrite from the assay medium. We describe a technique which allowed estimation of the enzyme level in leaf tissues of Vigna mungo (L). Hepper in terms of the release of the product (NH₃) of the enzyme reaction. The technique is offered as an alternative, possibly more convenient method for assay of nitrite reductase in plant tissue in vivo.     

Isolation and characterization of a unique protease from sporulating cells of Bacillus subtilis

Two proteases, designated I and II, have been isolated from sporulating cells of Bacillus subtilis. They were partially purified by ammonium sulfate fractionation, Sephadex chromatography and affinity columns. Protease I was found to be similar to an already characterized B. subtilis protease. Protease II is trypsin-like in its substrate specificity and is distinct from protease I in its pH optimum, pH stability, molecular weight, substrate specificity, heat stability and sensitivity to various inhibitors. While both enzymes were produced primarily during sporulation, they attained maximum levels of activity at different times. Distinct functions for these proteases in post exponential B. subtilis are likely.     

A study of the pollen grains of Amaranthus spinosus Linné and A. Dubius Mart ex Thellung and their hybrids

A pollen-morphology study of Amaranthus spinosus, A. dubius, and their hybrids has been carried out. Three pollen types have been observed, namely (1) Type A: micrograins; (2) Type B: grains with smaller pores; and (3) Type C: grains with larger pores. Type B is characteristic of A. spinosus, Type C of A. dubius, and the micrograins of the hybrids. Pollen size range, and frequency of the various morphotypes serve to throw light on the biosystematics of the plants studied.     

Purification and properties of pea cotyledon and embryo diamine oxidase

Diamine oxidase was purified separately from cotyledon and embryo of pea seedlings germinated for 6 days. The K_m of the cotyledon enzyme for putrescine was 1.6 × 10^?4M while that for the embryo enzyme was 9 × 10^?5M. On heating for 15 min at 70° the embryo enzyme retained about 90% activity whereas the cotyledon enzyme retained only 20% activity. The electrophoretic mobility of the cotyledon enzyme was ca twice that of the enzyme from embryo.     

Effect of ethrel and chloroethanol on pea diamine oxidase activity

The activity of cotyledon and embryo diamine oxidase was reduced by feeding ethrel and chloroethanol to the seedlings. The inhibitory effect of 2,4-D on the activity of enzyme in the cotyledon which may be mediated through ethylene was reversed by exposure of seeds to red light.