Generation of murine stromal cell lines supporting hematopoietic stem cell proliferation by use of recombinant retrovirus vectors encoding simian virus 40 large T antigen.

The bone marrow is a complex microenvironment made up of multiple cell types which appears to play an important role in the maintenance of hematopoietic stem cell self-renewal and proliferation. We used murine long-term marrow cultures and a defective recombinant retrovirus vector containing the simian virus 40 large T antigen to immortalize marrow stromal cells which can support hematopoiesis in vitro for up to 5 weeks. Such cloned cell lines differentially supported stem cells which, when transplanted, allowed survival of lethally irradiated mice, formed hematopoietic spleen colonies in vivo, and stimulated lymphocyte proliferation in vitro. Molecular and functional analyses of these cell lines did not demonstrate the production of any growth factors known to support the proliferation of primitive hematopoietic stem cells. All cell lines examined produced macrophage colony-stimulating factor. The use of immortalizing retrovirus vectors may allow determination of unique cellular proteins important in hematopoietic stem cell proliferation by the systematic comparison of stromal cells derived from a variety of murine tissues.     

Lack of cardiovascular tolerance during intravenous cocaine infusions in human volunteers

Acute tolerance to the cardiovascular effects of cocaine has been hypothesized from experiments in which the plasma concentrations of cocaine were rapidly changing. We studied the cardiovascular responses of 8 male human subjects for 4 hours following intravenous bolus doses of cocaine, and compared these to responses in the same subjects after intravenous bolus doses of cocaine followed by continuous intravenous infusions of cocaine designed to maintain steady state plasma levels of cocaine. We found little evidence of tolerance to the tachycardia and hypertensive effects of cocaine during a four hour exposure. Lack of tolerance to the cardiovascular effects of cocaine may be a factor in some types of cocaine related toxicity among cocaine abusers.     

High efficiency gene transfer into murine T cell clones using a retroviral vector

To establish a gene transfer and expression system for murine T cell clones, we have introduced the neomycin phosphotransferase gene encoding resistance to the neomycin analogue, G418, into non-neoplastic inducer T cell clones by using a replication-defective retroviral vector. This method allowed highly efficient gene transfer (20 to 40%) into two inducer T cell clones. The level of viral RNA expression in G418r T cells was 0.1% of poly(A)+ RNA. The infected G418r cells retained physiologic responsiveness to specific antigen as judged by antigen-specific proliferation and production of IL 3.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

The Rat Electroretinogram : II. Bloch's law and the latency mechanism of the b-wave

Electroretinograms were obtained from the all-rod eye of the rat with uniform illumination of the entire retina and stimulus flashes of less than 3 msec. duration. Bloch's law of temporal summation was verified for the b-wave latency by varying the time between two equal intensity flashes and observing that no change occurred in the latency when measured from the midpoint of the two flashes. The results of this and other experiments are described in terms of a simple but general model of the latency-determining mechanism. It is shown that this latency mechanism acts as if it depends on a linear additive process; and also that a hypothetical excitatory substance which triggers activity in the sources of the b-wave must accumulate rapidly in time after the flash, approximately as t⁸. The rate at which this substance accumulates is accurately represented by the diffusion equation for more than 4 to 6 log units in the flash intensity. This suggests that the rate-determining step in the latency mechanism may be diffusion-limited.     

Evidence for more than one Ia antigenic specificity on molecules determined by the I-A subregion of the mouse major histocompatibility complex.

Ia antigenic specificities determined by the I-A subregion of the mouse major histocompatibility complex have been examined in strain B10.D2 (H-2d), C57BL/10 (H-2b), and in a (C57BL/6xDBA/2) hybrid (BDF1; H-2b/d). Detergent solubilized, 3H-leucine-labeled antigen preparations were mixed with appropriate alloantisera and precipitation was induced either by addition of goat anti-mouse gamma-globulin or by addition of protein A-bearing Staphylococci. Sequential precipitation analysis showed that in strain B10.D2, Ia specificities 8 and 11 were co-precipitable, and that in strain C57BL/10, Ia specificities 8 and 9 were co-precipitable. In contrast, precipitation of specificities 9 and 11 from a BDF1 antigen preparation showed that these two Ia specificities were on separate molecules. The genetic implications of these data are discussed.     

The electric dipole moment of rhodopsin solubilized in Triton X-100.

The electric dipole moment of solubilized rhodopsin was determined with dielectric dispersion measurements. Rhodopsin was extracted from disc membranes of cattle rod outer segments with the nonionic detergent Triton X-100. The dipole moment of rhodopsin at its isoionic point in the detergent micelle is 720 D (150 charge-A). This value is comparable to dipole moments of nonmembrane proteins, especially those which tend to aggregate or polymerize. Flash irradiation of the rhodopsin results in an increase in the dipole moment of about 25 D (5 charge-A). The light-induced increase in dipole moment appears to be composed of two parts--a faster component related to a change in the number of protons bound by rhodopsin and a slower component apparently independent of the change in proton binding.     

Zinc and cadmium hyperaccumulation by Thlaspi caerulescens from metalliferous and nonmetalliferous sites in the Mediterranean area: implications for phytoremediation

Growth, tolerance and zinc and cadmium hyperaccumulation of Thlaspi caerulescens populations from three metal contaminated soils and three normal soils were compared under controlled conditions. Individuals of six populations were cultivated on five soils with increasing concentrations of zinc (50–25000 μg g⁻¹) and cadmium (1–170 μg g⁻¹). There was no mortality of normal soil populations in the four metal-contaminated soils, but plant growth was reduced to half that of populations from metal-contaminated soils. However, in noncontaminated soil, the growth of individuals from normal soils was greater than that of individuals from metal-contaminated soils. Individuals from normal soils concentrated three times more zinc in the aboveground biomass than those from metal-contaminated soils, but the latter accumulated twice as much cadmium. We conclude that populations of T. caerulescens from both normal and metal-contaminated soils are interesting material for phytoextraction of zinc and cadmium, but to optimize the process of phytoextraction it is necessary to combine the extraction potentials of both type of populations.