Recovery of the simian immunodeficiency virus (SIV) and depression of colony formation in in vitro infected progenitor cell-enriched rhesus bone marrow (BM)

Rhesus progenitor-enriched BM was exposed overnight to SIV and cultured in a limiting dilution assay where the potential for progenitor interaction with lymphocytes or macrophages was low. Virus was consistently isolated late in culture, detection being aided by coculture with CEM₁₇₄ lymphoblasts. Although infected cells had reduced clonogenic activity, colonies were indistinguishable from those derived from uninfected BM with respect to proliferative potential, morphology, and longevity in culture. Primate immunodeficiency viruses, therefore, may infect immature BM populations, directly affecting hematopoietic activity.     

A receptor for protein import into potato mitochondria

Five potential surface receptors for protein import into plant mitochondria were identified by gentle trypsin treatment of intact mitochondria from potato tubers and subsequent preparation of outer mitochondrial membranes. One of them, a 23 kDa protein, was purified to homogeneity and analysed by direct protein sequencing. Copy DNA clones encoding the corresponding polypeptide were isolated with labelled oligonucleotides derived from the amino acid data. The 23 kDa protein shares significant sequence similarity with protein import receptors from fungal mitochondria and contains one of their typical tetratricopeptide motifs. Its integration into the outer membrane is independent of protease accessible surface receptors and not accompanied by proteolytic processing. Monospecific antibodies against the 23 kDa protein significantly reduce import capacity of isolated mitochondria indicating that this component is indeed involved in the recognition or import of precursor proteins. As in fungi, immunological inhibition of protein import with IgGs against a single receptor is incomplete suggesting the existence of other receptors in the outer mitochondrial membrane of plant mitochondria.     

Development of a new method for obtaining osteoclasts from endosteal surfaces

Summary Techniques for the isolation of ahhighly pure population of viable osteoclasts are limited. For this reason, we developed an isolation procedure that results in a high yield of osteoclast-like cells, up to 92% pure, from 3-wk-old chicken tibias. The unique feature of the method is the migration of cells from marrow-free endosteal surfaces to vitronectin-coated plates. The cells retain the osteoclast phenotype and remain viable in culture for a minimum of 1 wk. The cells were characterized and compared to two populations of authentic avian osteoclasts, which were isolated on the basis of association with fibronectin-coated plates. The cells contained substantial amounts of tartrate-resistant acid phosphatase. Alkaline phosphatase levels were negligible, suggesting little contamination by osteoblasts. Response to parathyroid hormone, dibutyryl cyclic adenosine monophosphate, calcitonin, acetazolamide, 17β-estradiol, and prostaglandin E₂ was evident, as detected by measuring acid production. The vitronectin-associating cells contained numerous mitochondria, had the ability to resorb bone in anin vitro bone slice assay, and specifically bound biotinylated vitronectin. At 5 d of culture, the cells demonstrated marginal multinuclearity, having two to three nuclei. A large number (∼1×10⁶ cells/tibia) of viable cells that exhibit characteristics of authentic osteoclasts can be obtained by the method described. Potentially, this method could be applied to other species.     

Sperm motility in the presence of boundaries

The fluid dynamics of sperm motility near both rigid and elastic walls is studied using the immersed boundary method. Simulations of both single and interacting organisms are presented. In particular, we find that nearby organisms originally undulating with a 90° phase shift may adjust their relative swimming velocities and phase-lock. Comparisons with previous analytical results are also discussed. The tendency of a near-wall to attract organisms is demonstrated.     

Repair mechanisms of UV-induced DNA damage in soybean chloroplasts

In order to better understand the biochemical mechanisms of DNA metabolism in chloroplasts, repair of UV induced plastome damage in vivo was determined by exposure of soybean suspension cells to UV light and subsequent quantitation of the damage remaining in nuclear and chloroplast encoded genes with time by quantitative polymerase chain reaction (QPCR). The kinetics of damage rapir in the nuclear rbcS gene suggest that photoreactivation and dark mechanisms are active, while for the plastome encoded psbA gene only a light-dependent repair process was detected which is considerably slower than would be expected for photolyase-mediated photoreactivation.     

Staphylococcus haemolyticus contains two D-glutamic acid biosynthetic activities, a glutamate racemase and a D-amino acid transaminase.

Two D-glutamic acid biosynthetic activities, glutamate racemase and D-amino acid transaminase, have been described previously for bacteria. To date, no bacterial species has been reported to possess both activities. Genetic complementation studies using Escherichia coli WM335, a D-glutamic acid auxotroph, and cloned chromosomal DNA fragments from Staphylococcus haemolyticus revealed two distinct DNA fragments containing open reading frames which, when present, allowed growth on medium without exogenous D-glutamic acid. Amino acid sequences of the two open reading frames derived from the DNA nucleotide sequences indicated extensive identity with the amino acid sequence of Pediococcus pentosaceous glutamate racemase in one case and with that of the D-amino acid transaminase of Bacillus spp. in the second case. Enzymatic assays of lysates of E. coli WM335 strains containing either the cloned staphylococcal racemase or transminase verified the identities of these activities. Subsequent DNA hybridization experiments indicated that Staphylococcus aureus, in addition to S. haemolyticus, contained homologous chromosomal DNA for each of these genes. These data suggest that S. haemolyticus, and probably S. aureus, contains genes for two D-glutamic acid biosynthetic activities, a glutamate racemase (dga gene) and a D-amino acid transaminase (dat gene).     

Phenotypic features of breast cancer cells overexpressing ornithine-decarboxylase

Polyamines (PA) have been shown to be critical mediators of estradiol-induced breast cancer cell proliferation. This finding suggests that constitutive activation of the PA pathway may promote tumor progression, possibly leading to hormone independence. To test this hypothesis, we transfected hormone-responsive MCF-7 breast cancer cells with a complementary DNA coding for ornithine-decarboxylase (ODC), the first rate-limiting enzyme in PA biosynthesis. Marked ODC over-expression observed in stably transfected clones was associated with a selective increase in cellular putrescine content, while spermidine and spermine levels were not altered. ODC-overexpressing MCF-7 cells were resistant to the antiproliferative effects of low but not high concentrations of the enzyme inhibitor, α-difluoromethylornithine. In agreement with our hypothesis, sensitivity to the growth-promoting action of estradiol was reduced by approximately one third (P < 0.001) in ODC-overexpressing MCF-7 cells compared with vector-only transfected clones. Basal growth under anchorage-dependent conditions was only marginally increased by ODC overexpression (P = 0.048), while clonogenicity in soft agar was actually reduced. These data suggest that activation of PA biosynthesis may contribute in part to the acquisition of estrogen independence by breast cancer cells. Since only putrescine content was increased as a result of ODC overexpression, these data may underestimate the overall influence of the PA pathway on breast cancer phenotype. © 1995 Wiley-Liss, Inc.     

AN ANALYSIS OF SELECTIONAL RESPONSE IN RELATION TO A POPULATION BOTTLENECK

Selection for increased morphometric shape (ratio of wing length to thorax width) was compared between control (nonbottlenecked) populations and bottlenecked populations founded with two male–female pairs of flies. Contrary to neutral expectation, selectional response was not reduced in bottlenecked populations, and the mean realized heritabilities and additive genetic variances were higher for the bottlenecked lines than for the nonbottlenecked lines. Additive genetic variances based on these realized heritabilities were consistent with independent estimates of genetic variances based on parent–offspring covariances. Joint scaling tests applied to the crosses between selected lines and their controls revealed significant nonadditive components of genetic variance in the ancestor, which were not detected in the crosses involving bottlenecked lines. The nonbottlenecked lines responded principally by changes in one trait or the other (wing length or thorax width) but not in both, and regardless of which trait responded, larger trait size was dominant and epistatic to smaller size. Stabilizing selection for morphometric shape in the ancestor likely molded the genetic architecture to include nonadditive genetic effects.     

Dobutamine as a countermeasure for reduced exercise performance of rats exposed to simulated microgravity

Tipton, Charles M., and Lisa A. Sebastian. Dobutamineas a countermeasure for reduced exercise performance of rats exposed tosimulated microgravity. J. Appl.Physiol. 82(5): 1607-1615, 1997.Post-spaceflightresults and findings from humans and rodents after conditions of bedrest or simulated microgravity indicate maximum exercise performance issignificantly compromised. However, the chronic administration ofdobutamine (a synthetic adrenomimetic) to humans in relevantexperiments improves exercise performance by mechanisms that preventthe decline in peak O₂ consumption (O_{2 peak}) and reducethe concentration of lactic acid measured in the blood. Althoughdobutamine restores maximumO₂values in animals participating in simulated microgravitystudies, it is unknown whether injections of this₁-,₁-, and₂-adrenoceptor agonist in ratswill enhance exercise performance. To investigate this, adult male ratswere assigned to three experimental groups: caged control receivingsaline; head-down, tail-suspended (HDS) receiving saline (HDS-S); andan HDS group receiving dobutamine hydrochloride injections (1.8 mg/kgtwice daily per rat). Treadmill tests were performed before suspension,at 14 days, and after 21 days.O_{2 peak}, run time,and the rate of rise in colonic temperature (heating index) wereevaluated after 14 days, whereas at 21 days, hemodynamic responses(heart rate, systolic blood pressure, and double product) weredetermined during submaximal exercise with blood pH, blood gases, andlactic acid concentration values obtained during maximal exercise. Incontrast to the results for the HDS-S rats, dobutamine administrationdid restore O_{2 peak} and "normalized" lactic acid concentrations during maximalexercise. However, daily injections were unable to enhance exerciseperformance aspects associated with treadmill run time, the mechanicalefficiency of running, the heating index, or the retention of muscleand body mass. These simulated microgravity findings suggest that dobutamine's potential value as a countermeasure for postflight maximal performance or for egress emergencies is limited and that othercountermeasures must be considered.