Bone strontium in pregnant and lactating females from archaeological samples

Because plants and animals consume or absorb different amounts of strontium and calcium, anthropologists are able to use strontium/calcium (Sr/Ca) ratios from archaeologically recovered human bone to estimate the relative contributions of meat and plants to paleodiets. Often females exhibit higher Sr/Ca ratios than males, a fact usually attributed to lower meat intake among women. However, in vivo and in vitro experiments with laboratory animals show that pregnancy and lactation elevate maternal bone strontium and depress maternal bone calcium because 1) strontium is discriminated against in favor of calcium in the transport of ions to the placenta and mammary glands and 2) pregnancy and lactation facilitate absorption of alkaline earth metals from the gut. In this study, bone Sr/Ca ratios and strontium concentrations were compared between reproductive-age females, postmenopausal females, and adult males from two late prehistoric Native American sites in Georgia: the King site (N = 43) and the Etowah site (N = 51). At the King site, the mean Sr/Ca ratio of females was over 14% greater than that of males. At Etowah, the mean strontium level of reproductive-age females exceeded that of postmenopausal females by almost 25%. Most of the difference, it is argued, is due to pregnancy and lactation. A dietary preference among pregnant and lactating women for foods high in alkaline earths, particularly nuts and corn, may also be partially responsible. Until we assess the influence variables other than nutrition exert on trace element concentrations, our reconstructions of paleodiets will be suspect.     

Elevated levels of erythrocyte hypoxanthine phosphoribosyltransferase associated with allelic variation of murine Hprt

Murine stocks with wild-derived hypoxanthine phosphoribosyltransferase (HPRT) A alleles (Hprt a) have erythrocyte HPRT activity levels that are approximately 25-fold (Mus musculus castaneus) and 70-fold (Mus spretus) higher than those of laboratory strains of mice with the common Hprt b allele (Mus musculus: C3H/HeHa or C57B1/6). Since the purified HPRT A and B enzymes have substantially similar maximal specific activities (64 and 46 units/mg of protein, respectively), we infer that these HPRT activity levels closely approximate the relative levels of HPRT protein in these cells. Red blood cells of HPRT A and B mice have similar levels of adenine phosphoribosyltransferase activity (APRT; EC 2.4.2.7) and reticulocyte percentages, which suggests that the elevated levels of HPRT in erythrocytes of HPRT A mice are not secondary consequences of abnormal erythroid cell development. The HPRT activity levels in reticulocytes of HPRT B mice are approximately 35-fold higher than the levels in their erythrocytes and approach the HPRT activity levels in reticulocytes of HPRT A mice. Thus, the marked differences in the levels of HPRT protein in erythrocytes of HPRT A and B mice result from differences in the extent to which the HPRT A and B proteins are retained as reticulocytes mature to erythrocytes. The substantial and preferential loss of HPRT B activity from reticulocytes is paralleled by an equivalent loss of HPRT immunoreactive protein (i.e., CRM) from that cell, and we infer that the HPRT B protein is degraded or extruded as reticulocytes mature to erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mammalian cells are not killed by DNA single-strand breaks caused by hydroxyl radicals from hydrogen peroxide

Cell killing by ionizing radiation has been shown to be caused by hydroxyl free radicals formed by water radiolysis. We have previously suggested that the killing is not caused by individual OH free radicals but by the interaction of volumes of high radical density with DNA to cause locally multiply damaged sites (LMDS) (J. F. Ward, Radiat. Res. 86, 185-195, 1985). Here we test this hypothesis using hydrogen peroxide as an alternate source of OH radicals. The route to OH production from H2O2 is expected to cause singly damaged sites rather than LMDS. Chinese hamster V79-171 cells were treated with H2O2 at varying concentrations for varying times at 0 degree C. DNA damage produced intracellularly was measured by alkaline elution and quantitated in terms of Gray-equivalent damage by comparing the rate of its elution with that of DNA from gamma-irradiated cells. The yield of DNA damage produced increases with increasing concentration of H2O2 and with time of exposure. H2O2 is efficient in producing single-strand breaks; treatment with 50 microM for 30 min produces damage equivalent to that formed by 10 Gy of gamma irradiation. In the presence of a hydroxyl radical scavenger, dimethyl sulfoxide (DMSO), the yield of damage decreases with increasing DMSO concentration consistent with the scavenging of hydroxyl radicals traveling an average of 15 A prior to reacting with the DNA. In contrast to DNA damage production, cell killing by H2O2 treatment at 0 degree C is inefficient. Concentrations of 5 X 10(-2) M H2O2 for 10 min are required to produce significant cell killing; the DNA damage yield from this treatment can be calculated to be equivalent to 6000 Gy of gamma irradiation. The conclusion drawn is that individual DNA damage sites are ineffectual in killing cells. Mechanisms are suggested for killing at 0 degree C at high concentrations and for the efficient cell killing by H2O2 at 37 degrees C at much lower concentrations.     

GROWTH AND ORGANIZED DEVELOPMENT OF CULTURED CELLS. V. THE GROWTH OF COLONIES FROM FREE CELLS ON NUTRIENT AGAR

When angiosperm cells are cultured in a liquid medium they may grow in the form of free, single cells and form small to large groups of cells. This has been shown in earlier papers. This paper deals with the growth of strains of cells (Daucus carota and Haplopappus gracilis cells were used), washed and filtered free from the larger groups, on nutrient agar media in Petri dishes, thus simulating familiar microbiological technique. Each discrete member of a suspension is referred to as a “unit.” On the order of 1–10% of the separate units of a suspension may be induced to grow into viable colonies, depending on the strain and the conditions employed. Whereas at least 30% of the free single carrot cells were shown to be capable of division, only up to about 4% continued their growth to form macroscopically visible colonies when they were widely dispersed. Coconut milk promotes the growth of carrot cells into colonies. Both coconut milk and napthaleneacetic acid, which interact synergistically, arc required for the growth of Haplopappus cells. Various techniques which affect viability (the frequency with which units grow into colonies) were investigated. The viability of carrot units was found (1) to increase with their density on the plates; (2) to decrease upon washing the suspensions prior to plating; (3) to increase with increasing initial size; and (4) to decrease to a vanishingly low value in rigorously filtered suspensions which consist principally of single cells, although the single cells were found to grow with appreciable frequency when the larger units were also present; and (5) to increase dramatically (100-fold) when a rigorously filtered suspension was plated on a medium upon which pieces of growing cultured tissue were placed. Thus, the induction of growth in free cells is enhanced, even in an environment nutritionally optimal for the growth of the larger cell masses, by as yet unknown factors which are contributed by the cells themselves, or by adjacent cells or groups of cells. It is suggested that within a group of cells growing in culture, and perhaps also in the organized growing regions of intact plants, the dividing cells are nourished or stimulated by adjacent but less frequently dividing cells. The implications of these results are discussed.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Protein synthesis modulates the biological effectiveness of the combined action of hyperthermia and high-LET radiation.

The combined effects of heavy-ion radiation and hyperthermia on the survival of CHO-SC1 cells and its temperature-sensitive (ts) mutant tsH1 cells were studied using accelerated neon ions followed by mild heating at 41.5 degrees C. The sequence of application of heat and high-LET radiation is significant to cell-killing effects. Heat applied to cells prior to irradiation with neon plateau ions (LET = 32 keV/microns) was less effective than heat applied immediately after irradiation. The ability of cells to synthesize new proteins plays a key role in this sequence-dependent thermal sensitization. When protein synthesis was shut down in tsH1 cells, the thermal enhancement of cell killing by high-LET radiation was the same regardless of the sequence. The thermal enhancement of radiation-induced cell killing was LET-dependent for the SC1 cells, but this was not clearly demonstrated in the tsH1 cells. Furthermore, the RBE of heated SC1 cells varied with LET and reached a maximum of greater than 3 at 80 keV/microns. In the absence of protein synthesis, the maximum RBE value was reduced to 2.6. These results suggest that the accumulation of cellular damage caused by exposure to densely ionizing particles with increasing LETs can be potentiated with active protein synthesis during postirradiation heat treatment.     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Membrane behavior of exocytic vesicles: II in fate of the trichocyst membranes in paramecium after induced trichocyst discharge

A specific exocytic process, the discharge of spindle trichocysts of paramecium caudatum was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,0000 of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for that length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. the entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for tetrahymena.     

Uniparental isodisomy of chromosome 2 causing MRPL44-related multisystem mitochondrial disease

Mutations in nuclear-encoded protein subunits of the mitochondrial ribosome are an increasingly recognised cause of oxidative phosphorylation system (OXPHOS) disorders. Among them, mutations in the MRPL44 gene, encoding a structural protein of the large subunit of the mitochondrial ribosome, have been identified in four patients with OXPHOS defects and early-onset hypertrophic cardiomyopathy with or without additional clinical features. A 23-year-old individual with cardiac and skeletal myopathy, neurological involvement, and combined deficiency of OXPHOS complexes in skeletal muscle was clinically and genetically investigated. Analysis of whole-exome sequencing data revealed a homozygous mutation in MRPL44 (c.467 T?>?G), which was not present in the biological father, and a region of homozygosity involving most of chromosome 2, raising the possibility of uniparental disomy. Short-tandem repeat and genome-wide SNP microarray analyses of the family trio confirmed complete maternal uniparental isodisomy of chromosome 2. Mitochondrial ribosome assembly and mitochondrial translation were assessed in patient derived-fibroblasts. These studies confirmed that c.467 T?>?G affects the stability or assembly of the large subunit of the mitochondrial ribosome, leading to impaired mitochondrial protein synthesis and decreased levels of multiple OXPHOS components. This study provides evidence of complete maternal uniparental isodisomy of chromosome 2 in a patient with MRPL44-related disease, and confirms that MRLP44 mutations cause a mitochondrial translation defect that may present as a multisystem disorder with neurological involvement.