Kinetics of unscheduled DNA synthesis in UV-irradiated chicken embryo fibroblasts

Unscheduled DNA synthesis induced by 254-nm UV radiation in chicken embryo fibroblasts was examined for 24 h following irradiation, while cells were kept in the dark. The effect on this repair process of a 2-4 h exposure to photoreactivating light immediately after UV was studied. Initial [3H]thymidine incorporation in the light-treated cells was only slightly different from that in cells not exposed to light, but a distinct difference in rate and cumulative amount of unscheduled DNA synthesis was seen several hours after irradiation. By varying the UV dose and the time allowed for photoreactivation, the amount of dimers (determined as sites sensitive to a M. luteus UV-endonuclease) and non-dimers could be changed. The results of these experiments suggest that excision repair of dimers, rather than non-dimer products, is responsible for the unscheduled DNA synthesis seen after UV irradiation.     

The influence of multiple inseminations and multiple foundresses on social evolution

The breeding biology of a population determines the way in which individuals are distributed within and between progeny groups and, thus, affects the genetic variation within and between these groups. The breeding biology of any organism can be characterized by the distribution of the numbers of mates of females, the apportionment of paternity among males, the distribution of the numbers of females reproducing in common nests, and the apportionment of total fecundity within a nest among founding females. In addition, the possibility of genetic correlations among mates or among founding females is an important consideration and will be addressed in a later paper. The influence of the breeding biology on social evolution was evaluated by deriving the necessary conditions for the spread of genes for social behaviors and the rate of spread of these genes for populations with different breeding biologies. The first step in this derivation is to demonstrate that selection for genes determining social behavior can be represented as the covariance between gene frequency and relative fitness. Secondly, it is shown that this covariance can be formally partitioned into within and between group components. Thirdly, each covariance component is shown to be equivalent to the product of a genetic variance and a coefficient of linear regression of relative fitness on gene frequency. Lastly, specific models for the genotype fitnesses and breeding biologies are assumed and the necessary conditions for the increase in the frequency of altruistic alleles are obtained. The theory illustrates that variation in the numbers of mates per female has less of an effect on the evolution of social behaviors than does variation in the numbers of reproductive females per nest. In addition, it points out that the harmonic mean number of mates per female or of females per nest is a more useful summary statistic for characterizing populations with respect to the expected degree of evolved sociality than is the arithmetic mean.     

Early mouse embryos produce and release factors with transforming growth factor activity

Summary Previous studies have shown that extracts from mouse embryos at mid and late stages of development contain factors that exhibit transforming growth factor activity. The work reported here demonstrates that cultured mouse embryos at significantly earlier stages of development produce and release factors that exhibit the characteristic property of transforming growth factors. Specifically, the data demonstrate that embryos cultured from the blastocyst stage in serum-containing medium or in serum-free medium release factors that promote the anchorage-independent growth of normal rat kidney fibroblasts. It is shown that these factors are produced and released by cells derived from the inner cell mass and by trophoblasts. The precise developmental stage when production of these factors first begins has not been determined but our findings suggest that these factors are produced by cell types associated with early postimplatation embryos. This work was supported by the Laboratory of Viral Carcinogenesis at the National Cancer Institute and by grants from the National Cancer Institute (CA-36727) and the University of Nebraska Medical Center (22-271-732). Editor's Statement This paper presents evidence that, in an in vitro assay system, early embryonic cells are capable of both synthesizing and secreting TGF-like growth factors, implicating the production of these factors in the events of early development. David W. Barnes     

Thy-1 antigen expression by murine high-proliferative capacity hematopoietic progenitor cells. I. Relation between sensitivity to depletion by Thy-1 antibody and stem cell generation potential

Hematopoietic stem cells of high proliferative potential such as the giant macrophage colony-forming cell HPP-CFC, were present in the marrow of mice treated with high dose 5-fluorouracil (5Fu) (150 mg/kg i.v.), whereas most committed granulocyte-macrophage progenitors, GM-CFU-C, were depleted. Enrichment of primitive stem cells in post 5-Fu bone marrow (5FuBM) was reflected in an enhanced capacity to proliferate in suspension cultures stimulated by the mixture of lymphokines present in Con A spleen-conditioned medium supernatant (Con A CM) when compared to normal bone marrow. The population of blast-like cells harvested at 5 days from suspension cultures of 5FuBM with Con A CM showed marked increases in stem cells GM-CFU-C and HPP-CFC. For this reason, 5FuBM was utilized to study the cell surface characteristics of putative pluripotential stem cells capable of giving rise to committed stem cells in suspension cultures. Treatment of 5FuBM (BDF1 mice) before suspension culture with a high concentration of either of two cytotoxic monoclonal antibodies directed against the Thy-1.2 surface antigen in the presence of rabbit complement reduced or abrogated the generation of stem cells HPP-CFC and GM-CFU-C in suspension cultures, even though the input content of HPP-CFC and GM-CFU-C in treated 5FuBM compared with control 5FuBM showed little reduction by the antibody plus complement treatment. The Thy-1+ cell required for generation of stem cells was not a T cell, because reconstitution of Thy-1.2-depleted 5FuBM with spleen nylon nonadherent (T) cells did not reconstitute the generation of stem cells, even though T cells did grow in the suspension cultures. In addition, depletion from 5FuBM of cells expressing Lyt-1 and Lyt-2 antigens, unambiguous markers of T cell-thymocyte differentiation, did not ablate the generation of HPP-CFC and GM-CFU-C. Rather, performance of Thy-1 cell depletion at lower efficiency, which still abrogated T cell function, ablated generation of HPP-CFC but did not affect the generation of GM-CFU-C. It was concluded that 5FuBM contains distinct Thy-1+ primitive stem cells expressing different amounts of Thy-1 antigen correlating with their respective generation potentials. Some of these Thy-1+ progenitor cells may be pluripotential.     

Synthesis, biodistribution, and autoradiography of radiolabeled S-2-(3-methylaminopropylamino)ethylphosphorothioic acid (WR-3689)

35S- and 3H-labeled S-2-(3-methylaminopropylamino)ethylphosphorothioic acid (WR-3689) have been synthesized in our laboratory and used to study organ and cellular level distribution in C3H/Km mice bearing RIF-1 tumors. Tissue biodistributions obtained with 35S-WR-3689 showed that blood levels peak at 15 min postinjection and decline gradually over 60 min. At 30 min after drug injection the highest uptake is in kidney and submandibular salivary gland, with lowest levels in brain and moderate to low levels in the RIF-1 tumor, comparable to levels in skin and muscle. High resolution diffusible substance autoradiography with 3H-WR-3689 reveals a homogenous distribution of label over cells in liver and lung and nonuniform distribution of silver grains over the cytoplasm of cells in the kidney cortex, parotid and submandibular salivary glands, and small intestine. There are no indications of preferential nuclear location of label from protective drug in any tissue. Correlations of biodistribution and autoradiography data with measures of radioprotection in different tissues will be useful in interpreting mechanisms of radioprotection with this phosphorothioate.     

Physical Assay and Growth Cycle Studies of a Defective Adeno-Satellite Virus

Electron microscopic particle counting of the defective adeno-satellite virus (ASV), by use of pseudoreplication and negative staining with phosphotungstic acid, was shown to be a reproducible quantitative assay procedure. Particles of satellite type 4 that were counted in fluids from infected cultures had the same morphology as particles that banded at a buoyant density of 1.43 g/cc in cesium chloride. Other satellite virus serotypes examined in the same manner had a buoyant density of 1.37 to 1.38 g/cc. A comparison of satellite titers obtained by complement fixation and by particle counting demonstrated that an increase in satellite particles resulted in a corresponding increase in CF titers; however, electron microscopy was at least 10 times more sensitive than complement fixation for detecting satellite virus. Growth cycle studies of satellite virus in cells co-infected with adenovirus, as assayed by particle counting, indicated that the kinetics of satellite virus production closely followed the kinetics of its helper adenovirus production, with an eclipse period of 12 to 16 hr. The eclipse period of the satellite remained the same when cultures were preinfected with satellite 24 hr prior to adenovirus inoculation. However, when cultures were infected with adenovirus 12 hr before satellite virus, the eclipse period of the satellite was shortened to between 4 and 6 hr. Thus, satellite virus replication seems dependent upon a relatively late event in the adenovirus replication cycle. When cells were co-infected with adenovirus and its defective satellite, the yield of adenovirus was markedly reduced from that obtained in cells singly infected with adenovirus.     

Work capacity during 30 days of bed rest with isotonic and isokinetic exercise training

The purpose was to test the hypothesis that twice daily, short-term, variable intensity isotonic and intermittent high-intensity isokinetic leg exercise would maintain peak O2 uptake (VO2) and muscular strength and endurance, respectively, at or near ambulatory control levels during 30 days of -6 degrees head-down bed rest (BR) deconditioning. Nineteen men (aged 32-42 yr) were divided into no exercise control (peak VO2 once/wk, n = 5), isokinetic (Lido ergometer, n = 7), and isotonic (Quinton ergometer, n = 7) groups. Exercise training was conducted in the supine position for two 30-min periods/day for 5 days/wk. Isotonic training was at 60-90% of peak VO2, and isokinetic training (knee flexion-extension) was at 100 degrees/s. Mean (+/- SE) changes (P less than 0.05) in peak VO2 (ml.m-1.kg-1) from ambulatory control to BR day 28 were 44 +/- 4 to 36 +/- 3, -18.2% (3.27-2.60 l/m) for no exercise, 39 +/- 4 to 40 +/- 3, +2.6% (3.13-3.14 l/min) for isotonic, and 44 +/- 3 to 40 +/- 2, -9.1% (3.24-2.90 l/min) for isokinetic. There were no significant changes in any groups in leg peak torque (right knee flexion or extension), leg mean total work, arm total peak torque, or arm mean total work. Mean energy costs for the isotonic and isokinetic exercise training were 446 kcal/h (18.8 +/- 1.6 ml.min-1.kg-1) and 214 kcal/h (8.9 +/- 0.5 ml.m-1.kg-1), respectively. Thus near-peak, variable intensity, isotonic leg exercise maintains peak VO2 during 30 days of BR, while this peak, intermittent, isokinetic leg exercise protocol does not.     

Human immunodeficiency virus pseudotypes with expanded cellular and species tropism.

One mechanism for expanding the cellular tropism of a virus is through the formation of phenotypically mixed particles or pseudotypes, a process commonly occurring during viral assembly in cells infected with two or more viruses. We report here that dual infection of cells with human immunodeficiency virus (HIV) and a murine amphotropic retrovirus leads to the production of HIV pseudotypes that have acquired the host range of the amphotropic retrovirus and are capable of infecting not only CD4- human cells but also mouse cells. The replication of the HIV pseudotypes in the various CD4- cells was determined by measuring the appearance of HIV antigens in the supernatants, by cocultivation of CD4+ CEM cells with the infected CD4- cells, and in some cases by assaying the culture supernatants directly for infectious virus. Of the cells tested, human foreskin fibroblasts were the best host cells, and by in situ cytohybridization, we were able to document that all cells in the culture were infected. In addition, the temporal appearance of HIV-specific proteins in the HIV pseudotype-infected fibroblasts was similar to that seen in CD4+ CEM cells. If the human fibroblasts were first infected with the amphotropic retrovirus, they demonstrated the property of superinfection exclusion and were resistant to subsequent infection by the HIV pseudotype. In other cell lines, including the human glioblastoma-derived cell line U373MG, HeLa cells, BALB/c mouse embryo cells, and SC-1 wild mouse cells, although the HIV pseudotype infection appeared to be less efficient, substantial amounts of HIV were nevertheless produced. These results indicate that the HIV (amphotropic retrovirus) pseudotypes may be useful for studying the molecular biology of HIV infections in a wide range of cells.