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.     

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.

     

Postfertilization causes of differential success of pollen donors in Erythronium grandiflorum (Liliaceae): nonrandom ovule abortion

Seed paternity in Erythronium grandiflorum does not fully reflect the proportion of pollen on the stigma. When two types of pollen are simultaneously applied to the stigma, outbred seeds are produced over inbred, and seeds from more distant donors are produced over seeds from donors nearby. I looked for postfertilization causes of these previously reported patterns of differential success of pollen donors. I simultaneously pollinated stigmas with pollen from two donors and observed ovule development through a window sliced in the ovary. Pollen donor pairs were self and cross, donors 1 and 100 m from the recipient, and two donors each 100 m from the recipient. Since one donor was always the alternate homozygote from the recipient at the malate dehydrogenase locus, I could determine the paternity of developing seeds. When it appeared that ovules were aborting, I removed them and determined their paternity using starch gel electrophoresis. Ovules fertilized by self pollen were more likely to abort than ovules fertilized by cross pollen, and ovules fertilized by nearby donors were more likely to abort than ovules fertilized by distant donors. Ovules fertilized by donors 100 m from the recipient were equally likely to abort. There was not a significant relationship between the proportion of ovules fertilized by a pollen donor and the probability of those fertilized ovules developing into seeds. There was no relationship between ovule position within a fruit and ovule abortion. I manipulated available resources by removing leaves and by permitting only one fruit to develop per plant. Decreasing the amount of resources increased the proportion of aborted ovules. Abortion of ovules of lesser quality appears to release resources that can then be used to develop other offspring.     

A de novo satellited short arm of the Y chromosome possibly resulting from an unstable translocation

A satellited long arm of the Y chromosome (Yqs) is considered a normal variation, whereas the presence of a satellite on the short arm of the Y (Yps) has never been described in the literature. A Yps chromosome could be clinically significant if the translocation resulting in Yps has relocated the testis-determining gene, SRY, to another chromosome. A carrier of such a translocation would therefore be at increased risk for having XX male and XY female offspring. Here we describe the first reported case of de novo Yps present in a phenotypically normal male. This Yps chromosome was positive for C-banding and nucleolus organizer region (NOR) staining and showed a hybridization signal for the -satellite sequence. Fluorescence in situ hybridization (FISH) analysis indicated that SRY was retained on the Yps and the translocation breakpoint on Yps was distal to the pseudoautosomal region. At prenatal diagnosis, a normal appearing Y chromosome was found in his son, and thus the satellite on Yps was lost during meiotic Xp-Yp pairing. This Yps chromosome was likely the product of an unstable translocation.     

Protein kinase C is regulated in vivo by three functionally distinct phosphorylations

Background: Protein kinase Cs are a family of enzymes that transduce the plethora of signals promoting lipid hydrolysis. Here, we show that protein kinase C must first be processed by three distinct phosphorylations before it is competent to respond to second messengers.Results We have identified the positions and functions of the in vivo phosphorylation sites of protein kinase C by mass spectrometry and peptide sequencing of native and phosphatase-treated kinase from the detergent-soluble fraction of cells. Specifically, the threonine at position 500 (T500) on the activation loop, and T641 and S660 on the carboxyl terminus of protein kinase C βII are phosphorylated in vivo. T500 and S660 are selectively dephosphorylated in vitro by protein phosphatase 2A to yield an enzyme that is still capable of lipid-dependent activation, whereas all three residues are dephosphorylated by protein phosphatase 1 to yield an inactive enzyme. Biochemical analysis reveals that protein kinase C autophosphorylates on S660, that autophosphorylation on S660 follows T641 autophosphorylation, that autophosphorylation on S660 is accompanied by the release of protein kinase C into the cytosol, and that T500 is not an autophosphorylation site.Conclusion Structural and biochemical analyses of native and phosphatase-treated protein kinase C indicate that protein kinase C is processed by three phosphorylations. Firstly, trans-phosphorylation on the activation loop (T500) renders it catalytically competent to autophosphorylate. Secondly, a subsequent autophosphorylation on the carboxyl terminus (T641) maintains catalytic competence. Thirdly, a second autophosphorylation on the carboxyl terminus (S660) regulates the enzyme's subcellular localization. The conservation of each of these residues (or an acidic residue) in conventional, novel and atypical protein kinase Cs underscores the essential role for each in regulating the protein kinase C family.