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.     

Codon usage in the vertebrate hemoglobins and its implications

A study of codon usage in vertebrate hemoglobins revealed an evolutionary trend toward elevated numbers of CpG codon boundary pairs in mammalian hemoglobin alpha genes. Selection for CpG codon boundaries countering the generally observed CpG suppression is strongly suggested by these data. These observations parallel recently published experimental results that indicate that constitutive expression of the human alpha-globin gene appears to be determined by regulatory information encoded within the structural gene. The possibility is raised that, in the absence of selection, CpG decay can be used to date the evolutionary origin of a mammalian alpha pseudogene from its active alpha gene.      

Evolution of crystallins: expression of lens-specific proteins in the blind mammals mole (Talpa europaea) and mole rat (Spalax ehrenbergi)

The mole (Talpa europaea; Insectivora) and the mole rat (Spalax ehrenbergi; Rodentia) both have degenerated eyes as a convergent adaptation to subterranean life. The rudimentary eye lenses of these blind mammals no longer function in a visual process. The crystallin genes, which display a lens-specific expression pattern, were studied in these blind mammals and in related species with normal eyes by hybridizing their genomic DNAs with probes obtained from cDNA clones for alpha A-, alpha B-, and beta Bp-crystallins from calf and gamma 3- crystallin from the rat. For all crystallin genes examined, the hybridization signals of mole and mole rat genomic DNA were comparable, respectively, with those of shrew and of rat and mouse, normal-vision representatives of the orders Insectivora and Rodentia. The expression of the crystallins at the protein level was tested by using antiserum specific for alpha-crystallin in immunofluorescence reactions on lens sections of mole and mole rat eyes and by using antisera against the beta- and gamma-crystallins on sections of the mole eye. All antisera gave positive fluorescence reactions exclusively with lens tissue of these blind mammals, indicating that the crystallins are still normally expressed despite the fact that these lenses have had no function in a visual process in these mammals for at least many million years. These findings apparently imply that some unknown selective advantage has conserved the crystallin genes and their expression after the loss of normal function of the lenses.      

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.     

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.      

A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways.

The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.     

Evidence for gene conversion in the amylase multigene family of Drosophila pseudoobscura

The alpha-amylase (Amy) multigene family in Drosophila pseudoobscura is located on the third chromosome, which is polymorphic for more than 40 inverted gene arrangements. The number of copies in this family ranges from one to three, depending on the arrangement in question. A previous study of the three Amy genes from the Standard (ST) arrangement suggested either that duplicated copies (Amy2 and Amy3) are functionally constrained or that they are undergoing gene conversion with Amy1. In order to elucidate further the pattern of molecular evolution in this family, we cloned and sequenced four additional Amy genes, two from the Santa Cruz (SC) and two from the Chiricahua (CH) gene arrangement. Of the two alternatives, only the hypothesis of gene conversion is supported by the sequence analysis. The homogenization effect of gene conversion has been strongest in SC, whose copies differ by only two nucleotides, less noticeable in ST, and negligible in the CH. Furthermore, the action of gene conversion is apparently localized, occurring only in the coding region. Interestingly, these results concur with the findings of other workers for the duplicated Amy genes in the Drosophila melanogaster group. Thus, the occurrence of gene conversion in the Amy multigene family seems to be a common feature in the Drosophila species studied so far.