Genetic and physiological parameters associated with cadmium toxicity in Drosophila melanogaster

Two strains of Drosophila melanogaster represent the extremes in resistance and sensitivity to the lethal effects of CdCl₂. The strain containing the mutations vermilion and brown (v; bw) and the strain Austin had LC₅₀'s of 3.3 and 1.3mm CdCl₂, respectively. The three major chromosomes from these two strains were assorted genetically into the six possible combinations. The measured LC₅₀'s for CdCl₂ for these six genotypes fell into two groups according to the X chromosome; those containing the X chromosome from v; bw had LC₅₀'s 0.5–1.0mm greater than those in which the X chromosome was from Austin. Since the parent strains differed by 2mm, we suggest that the X chromosome is a major, but not the sole, site of genes to produce resistance to CdCl₂. When ¹⁰⁹Cd was in the diet the uptake by v; bw and Austin over 2 days was the same. After 4 days of uptake, the Austin strain excreted the ¹⁰⁹Cd five times faster than v; bw but the six genotypes did not differ appreciably in excretion rate from one another and resembled the sensitive parent Austin more than the resistant one. Thus a second process is indicated that distinguishes resistance to CdCl₂ that apparently is not associated with the X chromosome.This research was sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-84OR21400 with Martin Marietta Energy Systems, Inc.     

Effect of spermidine on the conformation of bacteriophage MS2 RNA. Electron microscopy and computer modeling

The structure of single-stranded RNA from the bacteriophage MS2 has been examined by electron microscopy in the presence of the polyamine spermidine. The molecules are found in two alternate conformations. The first of these can be characterized as a cruciform structure composed of three large loops approximately 500 to 700 nucleotides in size. The interior of the molecule has extensive base-paired regions which connect distant regions of the molecule; the farthest being 2500 nucleotides apart. In the second conformation, the molecules appear rod-like. Two of the large loops disappear, and these regions form, instead, extensive long-range helices. Computer modeling has been employed to explore the base-pairing potential of the sequence of bacteriophage MS2 RNA. Double-stranded regions identified by electron microscopy are shown to occur in local G + C-rich stretches of the RNA. Detailed models have been calculated for two regions of long-range contact. One of these includes the ribosome-binding site for the viral coat protein gene. The results are discussed in the context of the known role of RNA structure in the regulation of viral gene expression.     

Augmentation of cholinergic-mediated amylase release by forskolin in mouse parotid gland

Cholinergic-mediated amylase release in mouse parotid acini was augmented by forskolin; the potency but not the maximal response to carbachol was altered. Amylase released by carbachol plus forskolin was dependent on extracellular calcium and was mimicked by the calcium ionophore, A23187 plus forskolin. Forskolin was also shown to enhance carbachol-stimulated 45Ca2+ uptake into isolated acini. Hydroxylamine, nitroprusside, and 8-bromo-c-GMP each in combination with forskolin mimicked the effects of carbachol plus forskolin on amylase release. In the presence of carbachol (10(-8)M) forskolin did not augment c-AMP levels. However, in the presence of carbachol (5 X 10(-7) M) or hydroxylamine (50 microM) forskolin did significantly augment c-AMP accumulation. These results suggest that calcium and c-GMP may mediate the augmentation of cholinergic-mediated amylase release by effects on c-AMP metabolism.     

Neurite outgrowth traced by means of horseradish peroxidase inherited from neuronal ancestral cells in frog embryos

Outgrowing neurites in Xenopus embryos were labeled with horseradish peroxidase which had been injected into a single blastomere at the 32-cell stage and had been inherited by all the descendants, including neurons. Neurite outgrowth was traced from labeled trigeminal ganglion cells and most or all types of neurons present in the spinal cord at embryonic stages 20-30: primary motoneurons, commissural, dorsal longitudinal, ventral longitudinal, and Rohon-Beard neurons. All types of nerve fibers grew by the most direct pathway, apparently without errors of initial outgrowth, pathway selection, or target selection. An initial transient phase of outgrowth of filopodial processes from neuronal cell bodies and shafts of short neurites was observed which disappeared after further elongation of the neurites. The first pioneer fibers grew out from all types in a 2-hr period, from stage 20 to 22, and these fibers arrived at the targets within 3.5 hr after initial outgrowth. Additional fibers grew later in contact with the pioneers to form fascicles. Nerve fibers elongated without branching until they neared or contacted their targets. The rate of elongation at 20 degrees C was 30-75 micron/hr. The rapid, unbranched, error-free initial outgrowth and elongation of neurites to their targets is discussed in relation to theories of development of nerve pathways.     

Reversible addition of bisulfite buffer to the cytidine ring system

The rate constants for the reversible addition of protons and sulfite to the 5,6 double bond of cytidine and 3-methylcytidine have been spectrophotometrically measured under conditions (25°C, μ = 1.0 ) where the deamination of 5,6-dihydrocytidine-6-sulfonate is minimal. Both the addition and the elimination of sulfite from the ring system are subject to general catalysis of proton transfer. For the reaction in either direction, plots of the pseudo-firstorder rate constants against increasing buffer concentration are biphasic and indicative of at least a two-step reaction pathway with both steps being subject to general acid-base catalysis. Kinetic hydrogen-deuterium isotope effects were measured for both buffer-catalyzed steps of sulfite elimination from 3-methyl-5,6-dihydrocytidine-6-sulfonate and sulfite addition to 3-methylcytidine. Both H₂O and D₂O were used as solvent. For both the addition and the elimination of SO₃²⁻ values of k₂^H/k₂^D were 6.3–7.1 and 2.3–2.6 at low and high imidazole buffer concentration, respectively. The large isotope effects values in the range of 6–7 can be attributed to rate-determining proton transfer to carbon-5 of the cytidine ring system. The smaller values are more likely caused by proton transfer to a electronegative atom such as the oxygen on carbon-2 of the cytidine ring. The equilibrium constants for bisulfite buffer addition to 3-methylcytidine and cytidine at 25°C, μ = 1.0 , pH 7.2, are 10.2 and 1.3 ⁻¹, respectively.     

Migration of peripheral T and B cells into the thymus of aging (NZB × SJL)F1 female mice

Aging NZB × SJL (NS) female mice provide a unique model of thymopathology characterized by the intrathymic accumulation of large numbers of mature T and B cells. The purpose of the present work was to examine the possibility that this phenomenon results from the invasion of the thymus by cells from the periphery. Lymphoid cells labeled with chromium-51 or indium-111 were injected into syngeneic recipients to study their patterns of in vivo migration. Lymph node (LN) or spleen cells were found to localize significantly (1–2% of injected radioactivity) into the thymus of 12-month-old NS females but not into that of young recipients or of old NS males. However, intrathymic localization of injected LN cells was observed in castrated NS males which exhibit the same thymopathology as NS females. Both radiolabeled T and B cells were found to enter the thymus of aged NS females but the latter cells about three times less efficiently than the former. Moreover, while thymocytes from young NS females were unable to recirculate to LN, those of old NS females showed increased LN-seeking capacity and part (1%) of them did migrate back into the thymus of old but not young NS females. In additional cell transfer experiments, the intrathymic migration of B cells into old NS females was further documented by using the antibody response to sheep erythrocytes as a tracer. Taken together, these observations indicate that the thymus of aging NS female mice is permeable to recirculating lymphocytes, suggesting that at least part of the mature T and B cells detected in this thymus, are migrants from the periphery.     

Evaluation of the intramolecular stacking of the fluorosulfonylbenzoyl derivatives of 1,N6-ethenoadenosine, adenosine, and guanosine

The differences in conformation in solution of fluorosulfonylbenzoyl nucleosides were analyzed by fluorescence and proton nuclear magnetic resonance spectroscopy. The quantum yield of 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine (5'-FSB epsilon A) in aqueous solution is low (? = 0.01) as compared to that of its parent nucleoside, ethenoadenosine (? = 0.54), and increases approximately 5-fold when measured in a series of solvents of decreasing dielectric constant. The quantum yield of 5'-p-sulfonylbenzoyl-1,N6-ethenoadenosine covalently bound to glutamate dehydrogenase and pyruvate kinase is also 0.01, suggesting that the analogue may exist in the same conformation when enzyme-bound as when free in solution. In D2O, the resonances of the purine ring protons on 5'-FSB epsilon A, 5'-p-fluorosulfonylbenzoyl adenosine (5'-FSBA), and 5'-p-fluorosulfonylbenzoyl guanosine (5'-FSBG) are shifted upfield by about 0.1-0.3 ppm relative to the corresponding protons of their parent nucleosides. The calculated difference in chemical shift (delta delta) decreases as the dielectric constant of the solvent decreases. The delta delta decreases with increasing temperature. These data indicate that 5'-FSB epsilon A, 5'-FSBA, and 5'-FSBG exist in aqueous solution in a conformation in which the purine ring is intramolecularly stacked with the benzoyl moiety. From the magnitude of change in delta delta for 5'-FSB epsilon A, 5'-FSBA, and 5'-FSBG as a function of solvent, it appears that the three analogues differ in their sensitivity to disruption of stacking. The solution conformation of these three fluorosulfonylbenzoyl nucleoside analogues may be an important determinant of their reaction with various enzymes and may explain differences among the analogues in their reaction with a single enzyme.     

Ontogeny of B lymphocyte function. XV. A role for thymus cells in the maturation of the capacity of a subpopulation of B cells to re-express surface immunoglobulin after treatment with anti-immunoglobulin

The maturation of the C57BL/6 B cell population to be able to re-express surface immunoglobulin (sIg) after its removal by treatment with rabbit antimouse Ig (RAMIg) was studied in a cell transfer system. It was found that thymus cells were required for the maturation of a subset of the B cell population to be able to re-express sIg. The B cell population of irradiated, thymectomized mice reconstituted with spleen cells from donors under 2 wk of age remained deficient in their ability to re-express sIg even after 4 wk residence in the cell transfer recipient. In contrast, if adult thymus cells were transferred together with the immature B cells, the B cell population matured to be able to re-express sIg after treatment with RAMIg. Approximately one-third of the B cell population appears to require thymus cells for this maturation. The maturation of the thymus cell population to be capable of mediating this maturation of the B cell population occurs in two steps: between 2 and 3 and between 3 and 4 wk of age. This timing corresponds to the age at which the B cell population of C57BL/6 mice normally acquires the capacity to re-express sIg, which we have previously shown to also occur in two steps. Thymus cells from 3-wk-old donors can mediate the first step in B cell maturation to be able to re-express sIg, but cannot mediate the second step in this maturation of the B cell population. Thymus cells from 4-wk-old donors can mediate both steps in the maturation of the B cell population. The results suggest that thymus cells are involved in regulating some aspects of B cell differentiation.     

Lateral diffusion of an 80,000-dalton glycoprotein in the plasma membrane of murine fibroblasts: relationships to cell structure and function

The lateral diffusion of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been measured. This antigen, identified through the use of monoclonal antibodies, is an integral glycoprotein distributed through the plasma membrane as judged by immunofluorescence and immunoelectron microscopy (see preceding paper). Measurements of fluorescence recovery after photobleaching were performed on the antigen-antibody complex within the plasma membrane of C3H/10T1/2 and NIH/3T3 cells after labeling the monoclonal antibody with fluorescein. Measurements were performed as a function of temperature, for interphase, mitotic, and G0 C3H/10T1/2 cells. The mean lateral diffusion coefficients (D) for the antibody-protein complex in interphase cells were in the range of 0.7-3.5 X 10(-10) cm2/s between 9 degrees and 37 degrees C, while that for the lipid analog probe, dihexadecylindocarbocyanine was about two orders of magnitude greater. This comparison indicates that peripheral interactions other than bilayer fluidity limit the lateral mobility of the antigen. The mobile fraction of mitotic, G0, and interphase cells showed a monotonic increase with temperature with most of the antibody-antigen complexes being free to move about 25 degrees C. Semi-quantitative interpretations of both the slow glycoprotein diffusion and the immobile fraction are offered. Comparison of diffusion coefficients for cells in different phases of the cell cycle does not reveal striking differences. Mobile fractions for G0 cells at 25 degrees C or less are substantially lower than in interphase cells. In all cases, there was a remarkably broad range of the fluorescence recovery data between different cells, resulting in up to a 10-fold variation in diffusion coefficients, which is far greater than the precision limits of the experiment. Diffusion values and mobile fractions were generally well within a factor of two when measured at several arbitrary points on a single cell. The origins of this cellular heterogenity remain to be elucidated. Lateral mobility in cell fragments and specific regions of single cells was also examined. The glycoprotein was mobile in ventral surface cell fragments. Its mobility was not altered in regions of cell- cell underlapping. However, the diffusion coefficient was threefold higher near the leading edge of motile cells compared to the trailing region. This difference may reflect weaker coupling of the glycoprotein to the underlying cytoskeleton in the dynamic leading edge region.     

Rearrangement of tubulin, actin, and myosin in cultured ventricular cardiomyocytes of the adult rat

Antitubulin, phalloidin, and antimyosin were used to study the distribution of microtubules, microfilaments, and myofibrils in cultured adult cardiomyocytes. These cells undergo a stereotypic sequence of morphological change in which myotypic features are lost and then reconstructed during a period of polymorphic growth. Microtubules, though rearranged during these events in culture, are always present in an organized network. Myosin and actin structures, on the other hand, initially degenerate. This initial degeneration is reversed when a cell attaches to the culture substratum. Upon attachment, new microtubules are laid down as a cortical network adjacent to the sarcolemma and, subsequently, as a network in the basal part of the cell. Actin and then myosin filament bundles appear next, in a pattern corresponding to the pattern of the microtubules. Finally, striated myofibrils are formed, first in the central part of the cell, and subsequently in the outgrowing processes of the cell. A mechanism is suggested by which the eventual polymorphic shape of a cell is related to the shape of its initial area of contact with the culture substratum. Finally, a model of myofibrillogenesis is proposed in which microtubules participate in the insertion of myosin among previously formed actin filament bundles to produce myofibrils.