Lectin-mediated uptake of lysosomal hydrolases by genetically deficient human fibroblasts.

A protein factor named S-II that stimulates RNA polymerase II was previously purified from Ehrlich ascites tumor cells [1]. In this work using an antibody prepared against purified S-II, the localization of S-II in the cell was investigated by an indirect immunofluorescence technique. In 3T3 cells, specific immunofluorescence was detected only in the nucleoplasm where RNA polymerase II is located, and not in the nucleoli where RNA polymerase I is present. In Ehrlich ascites tumor cells fluorescence was detected mainly in the nucleoplasm, although some fluorescence was also detectable in the cytoplasm, possibly due to leak of S-II from the nuclei during preparation of the immunofluorescent samples. In metaphase cells fluorescent was not found on chromosomes but throughout the cytoplasm. These findings suggest that S-II is a nuclear protein and that it spreads into the cytoplasm without being attached to chromosomes in metaphase, but is reassembled into the nucleoplasm in the interphase. Specific immunofluorescence was also detected in the nuclei of HeLa cells and salivary glands cells of flesh-fly larvae, suggesting that the nucleoplasm of these heterologous cells contains proteins immunologically cross-reactive with the antibody against S-II.     

Density distribution analysis of in vivo and in vitro colony forming cells in developing fetal liver

The technique of buoyant density separation in gradients of Bovine Serum Albumin has been used to separate in vivo and in vitro colony forming cells (C.F.C.'s) in hemopoietic tissue of mouse fetal liver. Differences in the density distribution profiles showed that the in vivo and in vitro C.F.C.'s were different cell populations but the existence of an “out-of-phase” density association suggested that the two cell types were closely related. Complex density heterogeneity of both cell populations was observed at later stages of liver development and was similar to that seen in adult marrow. A homogeneous population of in vivo and in vitro C.F.C.'s occupied a very light density position in 10.5 day fetal liver. The subsequent development of density heterogeneity was associated with progressive acquisition of higher density subpopulations. Transfer experiments showed the capacity of the lightest density cells from the earliest stage of liver hemopoiesis, to generate higher density colony forming cells in the environment of the adult marrow. Density determined differences in seeding efficiency of in vivo C.F.C.'s were observed but no evidence was obtained for differences in either in vivo or in vitro colony morphology in different density subpopulations.     

Purification, crystallization and preliminary crystallographic analysis of porcine aldose reductase

Large crystals of porcine aldose reductase have been grown from polyethylene glycol solutions. The crystals are triclinic, space-group P1, with a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees and gamma = 79.0 degrees. The crystals grow within ten days to dimensions of 0.6 mm x 0.4 mm x 0.2 mm and diffract to at least 2.5 A. There are four molecules in the unit cell related by a set of three mutually perpendicular non-crystallographic 2-fold axes.     

Inter- and intraclade neutralization of human immunodeficiency virus type 1: genetic clades do not correspond to neutralization serotypes but partially correspond to gp120 antigenic serotypes.

We have studied genetic variation among clades A through E of human immunodeficiency virus type 1 (HIV-1) at the levels of antibody binding to gp120 molecules and virus neutralization. We are unable to identify neutralization serotypes that correspond to the genetic clades. Instead, we observe that inter- and intraclade neutralization of primary isolates by HIV-1-positive sera is generally weak and sporadic; some sera show a reasonable degree of neutralization breadth and potency whereas others are relatively sensitive to neutralization, but no consistent pattern was found. However, a few sera were able to neutralize across clades with significant potency, an observation which may have implications for the feasibility of a broadly effective HIV-1 vaccine involving humoral immunity. Serological assays measuring anti-gp120 antibody binding also failed to identify serotypes that correspond precisely to the genetic clades, but some indications of clade-specific binding were observed, notably with sera from clades B and E. A representative protein for each clade (A through E) was selected on the basis of its specificity, defined as high seroreactivity with sera from individuals infected with virus of that clade and lower reactivity with sera from individuals infected with viruses from other clades. The seroreactivity patterns against these five proteins could be used to predict the genotype of the infecting virus with moderate success.     

Disrupted-In-Schizophrenia 1 regulates integration of newly generated neurons in the adult brain

Adult neurogenesis occurs throughout life in discrete regions of the adult mammalian brain. Little is known about the mechanism governing the sequential developmental process that leads to integration of new neurons from adult neural stem cells into the existing circuitry. Here, we investigated roles of Disrupted-In-Schizophrenia 1 (DISC1), a schizophrenia susceptibility gene, in adult hippocampal neurogenesis. Unexpectedly, downregulation of DISC1 leads to accelerated neuronal integration, resulting in aberrant morphological development and mispositioning of new dentate granule cells in a cell-autonomous fashion. Functionally, newborn neurons with DISC1 knockdown exhibit enhanced excitability and accelerated dendritic development and synapse formation. Furthermore, DISC1 cooperates with its binding partner NDEL1 in regulating adult neurogenesis. Taken together, our study identifies DISC1 as a key regulator that orchestrates the tempo of functional neuronal integration in the adult brain and demonstrates essential roles of a susceptibility gene for major mental illness in neuronal development, including adult neurogenesis.     

An antibody raised against in vitro-derived human mast cells identifies mature mast cells and a population of cells that are Fc epsilon RI(+), tryptase(-), and chymase(-) in a variety of human tissues.

Selective markers for human mast cells are of paramount importance for understanding their role in physiological and pathological processes. A mouse monoclonal antibody (MAb) designated 2C7, raised against in vitro-derived human mast cells, was used in immunoenzymatic analysis of sections from a variety of human organs. Double immunolabeling with 2C7 and tryptase, chymase, Fc epsilon RIalpha, and c-kit was performed on cryostat tissue sections from skin, colon, uterus, breast, stomach, bladder, and lung. MAb 2C7 stained greater than 93% of the tryptase(+) or chymase(+) mast cells in all tissues examined. In addition, the majority of cells stained with the tryptase or chymase also stained for Fc epsilon RIalpha. However, there were a significant number of Fc epsilon RIalpha(1) cells in all tissues studied that were tryptase(-) and/or chymase(-). In contrast, MAb 2C7 in double immunoenzymatic staining co-localized with 93-96% of the Fc epsilon RIalpha(1) cells in all tissues. Analysis for c-kit expression on the different tissues revealed that the majority of tryptase(+) or chymase(+) cells in skin, uterus, bladder, and lung stained with c-kit. However, only approximately 70-78% of tryptase(+) cells in colon and stomach were c-kit(+). These data suggest that MAb 2C7 appears to identify mature mast cells and a population of Fc epsilon RIalpha(1), chymase(-), and tryptase(-) cells in a variety of human tissues.     

Expression vectors for human adenosine deaminase gene therapy

Somatic gene transfer offers a possible new approach for treatment of human genetic disease. Defects affecting blood-forming tissues are candidates for therapies involving transfer of genetic information into hematopoietic stem cells. Adenosine deaminase (ADA) deficiency is being used as a model disease for which gene transfer techniques can be developed and evaluated. We describe here the construction and testing of 20 retroviral vectors for their ability to transfer and express human ADA in vitro and in vivo via a mouse bone marrow transplantation model. After infection of primary bone marrow with one fo these vectors (p delta NN2ADA), human ADA was detected in 60-85% of spleen colonies at day 14 and maintained long term in the blood of fully reconstituted mice. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.     

Double or Nothing: A Drosophila Mutation Affecting Meiotic Chromosome Segregation in Both Females and Males

We describe a Drosophila mutation, Double or nothing (Dub), that causes meiotic nondisjunction in a conditional, dominant manner. Previously isolated mutations in Drosophila specifically affect meiosis either in females or males, with the exception of the mei-S332 and ord genes which are required for proper sister-chromatid cohesion. Dub is unusual in that it causes aberrant chromosome segregation almost exclusively in meiosis I in both sexes. In Dub mutant females both nonexchange and exchange chromosomes undergo nondisjunction, but the effect of Dub on nonexchange chromosomes is more pronounced. Dub reduces recombination levels slightly. Multiple nondisjoined chromosomes frequently cosegregate to the same pole. Dub results in nondisjunction of all chromosomes in meiosis I of males, although the levels are lower than in females. When homozygous, Dub is a conditional lethal allele and exhibits phenotypes consistent with cell death.