Role of arginine 180 and glutamic acid 177 of ricin toxin A chain in enzymatic inactivation of ribosomes.

The gene for ricin toxin A chain was modified by site-specific mutagenesis to change arginine 180 to alanine, glutamine, methionine, lysine, or histidine. Separately, glutamic acid 177 was changed to alanine and glutamic acid 208 was changed to aspartic acid. Both the wild-type and mutant proteins were expressed in Escherichia coli and, when soluble, purified and tested quantitatively for enzyme activity. A positive charge at position 180 was found necessary for solubility of the protein and for enzyme activity. Similarly, a negative charge with a proper geometry in the vicinity of position 177 was critical for ricin toxin A chain catalysis. When glutamic acid 177 was converted to alanine, nearby glutamic acid 208 could largely substitute for it. This observation provided valuable structural information concerning the nature of second-site mutations.     

Intercellular structure in a many-celled magnetotactic prokaryote

A many-called magnetotactic prokaryote obtained from brackish water was observed to possess intercellular connections at points of contact between the outer membranes of constituent cells. Each aggregate organism consisted of 10 to 30 individual Gram-negative cells containing material with the appearance of poly--hydroxybutyrate and magnetosomes of unusual arrangement, structure and composition. The aggregate, which possessed prokaryotic-type flagella arranged at the outwards surfaces of each cell, showed motility indicative of co-ordination between individual component cells. These results suggest that this organism could be a multicellular prokaryote.     

Cellular uptake of the tat protein from human immunodeficiency virus

While developing an assay to measure the activity of the tat protein from human immunodeficiency virus 1 (HIV-1), we discovered that the purified protein could be taken up by cells growing in tissue culture and subsequently trans-activate the viral promoter. Trans-activation is dramatically increased by a variety of lysosomotrophic agents. For example, trans-activation can be detected at tat concentrations as low as 1 nM in the presence of chloroquine. Experiments using radioactive protein show that tat becomes localized to the nucleus after uptake and suggest that chloroquine protects tat from proteolytic degradation. These results raise the possibility that, under some conditions, tat might act as a viral growth factor to stimulate viral replication in latently infected cells or alter expression of cellular genes.     

Magnetic properties of tunicate blood cells. I. Ascidia nigra

The magnetic properties of intact and freeze-dried blood cells of the tunicate Ascidia nigra and of model vanadium(III) and (IV) compounds as polycrystalline solids and in aqueous solution have been measured up to 50 kOe with a SQUID susceptometer. Corrections for the samples' diamagnetism were extracted from the temperature dependence of the data without any further assumptions. For vanadium(IV), measured values of the magnetic moment at different values of the applied magnetic field over the temperature range 2-100 K obey a Brillouin function with spin 1/2. For vanadium(III), the magnetic moment data did not obey a Brillouin function and were analyzed in terms of a spin Hamiltonian with S = 1. Measurements on both whole and freeze-dried blood samples give consistent results with vanadium(III) the predominant species. These results are discussed in terms of the mechanisms of vanadium accumulation and the use of vanadium oxidation states as criteria of ascidian taxonomy.     

Prochlorococcus marinus nov. gen. nov. sp.: an oxyphototrophic marine prokaryote containing divinyl chlorophyll a and b

Several years ago, prochlorophyte picoplankton were discovered in the N. Atlantic. They have since been found to be abundant within the euphotic zone of the world's tropical and temperate oceans. The cells are extremely small, lack phycobiliproteins, and contain divinyl chlorophyll a and b as their primary photosynthetic pigments. Phylogenies constructed from DNA sequence data indicate that these cells are more closely related to a cluster of marine cyanobacteria than to their prochlorophyte relatives Prochlorothrix and Prochloron. Several strains of this organism have recently been brought into culture, and herewith are given the name Prochlorococcus marinus.     

Characterization of endogenous and recombinant proviral elements of a highly tumorigenic AKR cell line

As an approach to evaluating the contribution of classes of endogenous viral sequences to leukemogenesis, a genomic library was prepared from the highly tumorigenic AKR SL12.3 cell line and screened for env-containing proviruses. An extensive battery of virus-derived probes and specific oligonucleotide probes were used to segregate 83 positive clones into related groups. The nonecotropic endogenous retroviruses were identified as members of the polytropic, modified polytropic, or xenotropic groups. At least three unique xenotropic proviruses were detected that differed from the published xenotropic sequence within a variable region of the 5' portion of env. Changes among the xenotropic proviruses included relative insertions and/or deletions that maintain an open reading frame and hence the potential to encode viable envelope gene products. Several recombinant viruses were also detected. Recombination was not random and primarily involved the formation of mink cell focus-inducing class I retroviruses via recombination between polytropic elements and ecotropic virus. One other recombinant was detected which contained ecotropic virus sequences in the 5' region encoding p15 of an otherwise xenotropic provirus. An interesting observation was the finding that certain clones contained more than one provirus within the average 20-kb cloned insert. This would not be expected if integration were totally random. The de novo recombinant proviruses identified here provide a series of potential candidates to be evaluated for their contribution to the tumorigencity of the SL12.3 cell line.     

The AXB and BXA set of recombinant inbred mouse strains

The recombinant inbred (RI) set of strains, AXB and BXA, derived from C57BL/6J and A/J, originally constructed and maintained at the University of California/San Diego, have been imported into The Jackson Laboratory and are now in the 29th to 59th generation of brother-sister matings. Genetic quality control testing with 45 proviral and 11 biochemical markers previously typed in this RI set indicated that five strains had been genetically contaminated sometime in the past, so these strains have been discarded. The correct and complete strain distribution patterns for 56 genetic markers are reported for the remaining RI strain set, which consists of 31 living strains and 8 extinct strains for which DNA is available. Two additional strains, AXB 12 and BXA 17, are living and may be added to the set pending further tests of genetic purity. The progenitors of this RI set differ in susceptibility to 27 infectious diseases as well as atherosclerosis, obesity, diabetes, cancer, cleft palate, and hydrocephalus. Thus, the AXB and BXA set of RI strains will be useful in the genetic analysis of several complex diseases.     

The estrogen-receptor complex is bound at unusual chromatin regions

The estrogen receptor of MCF-7 cells labeled with high specific activity estradiol was used to mark the chromatin binding sites for this regulatory molecule. Many of these sites are especially sensitive to nuclease, and produce on digestion a series of uniquely sedimenting products. Several of these have been examined in some detail in this paper. These include a form of receptor that sediments in trace digests at 9S but in more extensive digests at 7S, fast mononucleosomes of about 12.5S, and a species at 15S. Two components of digests, fast mononucleosomes and dinucleosomes were isolated and subjected to further digestion. Much of the hormone on these isolated particles was found to be sensitive to additional hydrolysis, although some was nuclease resistant. It appears that a major fraction of the hormone receptor complexes bound to MCF-7 cell chromatin occurs at nucleosome-free regions which can be detected as transient hydrolysis intermediates.     

Hypophysectomy and hemivasectomy can inhibit the testicular hemicastration response of the mature rat

Three questions were asked in an attempt to understand how testosterone (T) concentration in the veins of the remaining testis can double within 24 h after hemicastration in the mature rat without a change in plasma luteinizing hormone (LH) levels. These three questions (and their answers) were: 1) Can the testicular hemicastration response occur in hypophysectomized rats? Answer, No. 2) Does LH binding to the testis increase after hemicastration? Answer, No. 3) Is there a neural route to the testis alternate to the superior spermatic plexi? Answer, Yes, apparently there is, since hemivasectomy contralateral to the excised testis partially suppressed the testicular hemicastration response (150.4 +/- 13.2 ng/ml in hemicastrated, sham- hemivasectomized rats [n = 18] vs. 109.4 +/- 11.6 ng/ml in hemicastrated, hemivasectomized rats [n = 18], P less than 0.026). It was concluded that LH was probably necessary to the testicular hemicastration response but that its presence did not provide a mechanism. The response was mediated at least partly through the inferior spermatic nerves associated with the vas deferens. A possible reason, although highly speculative, for failure to previously block the testicular hemicastration response by bilateral denervation of the superior spermatic plexi (Mock and Frankel , 1982) was that during the 12-wk interval between denervation and hemicastration, testicular innervation functionally transferred from the superior spermatic to the inferior spermatic nerves.