INTERACTION OF IONIZING AND VISIBLE RADIATION IN MUTATION INDUCTION IN NEUROSPORA CRASSA

Klein , Richard M. (New York Bot. Gdn., New York, N.Y.), and Deana T. Klein. Interaction of ionizing and visible radiation in mutation induction in Neurospora crassa. Amer. Jour. Bot. 49(8): 870–874. 1962.—Conidia of the purple adenineless strain of N. crassa were irradiated with 25 kr of X rays and then exposed to far-red or red radiations or to far-red followed by red radiation. Far-red light, without effect on un-irradiated conidia, augmented the genetic damage caused by X rays as measured by survival (colony count), back mutation to adenine prototrophy, and the induction of mutants affecting colony morphology. Post-X-irradiation with red light ameliorated the severity of X-radiation as measured by survival and back mutation. The potentiation of X-ray-induced genetic damage by far-red light could be completely negated by subsequent exposure to red light.     

Further purification and characterization of the succinyl-CoA:3-hydroxy-3-methylglutarate coenzyme A transferase from rat-liver mitochondria

Succinyl-CoA:3-hydroxy-3-methylglutarate coenzyme A transferase, previously identified in rat-liver mitochondria (Deana et al. (1981), Biochim. Biophys. Acta 662, 119-124), was purified to near homogeneity and further characterized. After the last purification steps consisting of Ultrogel AcA-44 filtration and agarose-hexane-coenzyme A chromatography, the enzyme was apparently tetrameric with a mass of 48-52 kDa determined by gel filtration on Sephadex G-75, ultracentrifugation through a sucrose gradient and SDS-gel electrophoresis. By means of a HPLC technique developed for measuring the CoA esters we could determine the enzyme activity in both forward and reverse directions and show that the kinetic constants, i.e., Km of reactants and Vmax, are not too different for the two reactions. Double-reciprocal plots of the enzyme velocities versus the concentration of one substrate at different fixed concentrations of the other substrate gave families of straight lines converging below the substrate-abscissa for both forward and backward reactions, indicating a kinetic mechanism of rapid equilibrium random Bi-Bi type. The competitive inhibition of the product succinate with respect to both reactants, 3-hydroxy-3-methylglutarate and succinyl-CoA, as well as the Haldane relationships are consistent with this conclusion. An inhibitory effect on CoA transferase activity by acetate, acetoacetate, acetyl-CoA, acetoacetyl-CoA, coenzyme A, carnitine, ZnCl2 and high concentrations of the monovalent anions ClO4-, F-, I- and Cl- was also found.     

Effects of calcium chelators, divalent cations and sulfhydryl reagents on calcium uptake and motility of bovine spermatozoa

Addition of 1 mM Ca/EGTA complex (1:1 ratio) to an incubation medium containing 1.5 mM Ca2+ produced a notable increase in the Ca2+ cycling in ejaculated bovine spermatozoa. Similar results were also obtained with the Ca/EDTA and Ca/EDTA complexes or with the heavy metal chelator DTPA (50 microM). Ba2+, Ni2+ or Co2+ added at 0.1 mM concentration abolished the stimulatory effect of the Ca/EGTA complex on Ca2+ cycling, whereas it did not affect the calcium movement in the absence of the calcium chelator complex. It is concluded that small amounts of these cations should be bound to the plasma membrane of bovine spermatozoa and inhibit the cellular calcium influx. 0.1 mM Cd2+ and NEM or 1 mM diamide produced a calcium efflux from the spermatozoa together with an inhibition of cellular motility and an increase in glutamate-oxaloacetate transaminase release. Conversely the impermeant sulfhydryl reagent mersalyl caused a net calcium efflux but did not alter the cellular motility nor the transaminase release. It is suggested that the permeant thiol reagents could decrease the spermatozoal mobility by impairing the mitochondrial ATP-synthesis.     

Characterization of a two-signal-dependent, Ia+ mononuclear phagocyte progenitor subpopulation that is sensitive to inhibition by ferritin

Evidence is presented that the ferritin-inhibitable, Ia+ monocyte progenitor in murine marrow requires two signals for stimulation of clonal proliferation. Escherichia coli K235 lipopolysaccharide (LPS) at 0.1 ng/ml enhanced macrophage colony formation by 25 to 70% in murine marrow cultures stimulated with colony-stimulating factor (CSF-1). The progenitors which responded to LPS and CSF-1 represented a distinct subpopulation. Pretreatment of marrow cells with complement plus anti-Ia, anti-H2, anti-asialo GM1, and anti-Mac-1 antibodies specifically depleted the two-signal-requiring progenitors. In addition, the same progenitors were depleted by preincubation with hydroxyurea, indicating that these cells were in cell cycle when removed from the marrow. When compared with the quiescent progenitors, the Ia+, cycling cells were more sensitive to the antiproliferative effects of interferon alpha/beta but were more resistant to inhibition by E prostaglandins. Pretreatment with T cell-specific antibodies and complement specifically enhanced cloning of quiescent progenitors without affecting cloning of the Ia+, cycling subpopulation. Moreover, rat liver ferritin at 10(-8) to 10(-10) M specifically inhibited clonal proliferation of the Ia+ progenitors. Finally, the requirement for LPS as the additional stimulant could be replaced by the addition of haplotype-specific anti-Ia antibody to CSF-stimulated cultures. In contrast to LPS, anti-IA was competitive with inhibitory ferritin in clonal proliferation of the Ia+ progenitors. The significance of these observations in regulation of monocytopoiesis is discussed.     

Platelet activation by diacylglycerol or ionomycin is inhibited by nitroprusside.

Experiments were performed to elucidate the role of cyclic guanosine monophosphate (cGMP) on platelet activation induced by protein kinase C (PKC) activators and calcium ionophore. Human platelets were pretreated with acetylsalicylic acid and with hirudin and apyrase. Aggregation and ATP secretion in response to the PKC activators 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl 2-acetylglycerol (OAG) were inhibited by the nitrovasodilator sodium nitroprusside (SNP), an activator of guanylate cyclase, and by 8-bromo-cyclic GMP (8-Br-cGMP). The experiments were performed in the presence of M&B 22948, an inhibitor of cGMP phosphodiesterase. SNP and 8-Br-cGMP also inhibited platelet aggregation and secretion evoked by the ionophore ionomycin. In fura-2 loaded platelets SNP did not affect basal cytosolic Ca2+ level nor the rise induced by low concentrations of ionomycin, both in the presence and absence of extracellular Ca2+. The phosphorylation of the 47 and 20 kDa protein induced by ionomycin or PMA were not significantly decreased by SNP or 8-Br-cGMP. The present results suggest that cGMP is able to inhibit both the PKC and the Ca(2+)-dependent pathways leading to platelet activation by interfering, similarly to cAMP, with processes following protein phosphorylation, close to the effector systems.     

Comparative analysis of quantitative trait loci controlling glucosinolates,myrosinase and insect resistance in Arabidopsis thaliana

Evolutionary interactions among insect herbivores and plant chemical defenses have generated systems where plant compounds have opposing fitness consequences for host plants, depending on attack by various insect herbivores. This interplay complicates understanding of fitness costs and benefits of plant chemical defenses. We are studying the role of the glucosinolate-myrosinase chemical defense system in protecting Arabidopsis thaliana from specialist and generalist insect herbivory. We used two Arabidopsis recombinant inbred populations in which we had previously mapped QTL controlling variation in the glucosinolate-myrosinase system. In this study we mapped QTL controlling resistance to specialist (Plutella xylostella) and generalist (Trichoplusia ni) herbivores. We identified a number of QTL that are specific to one herbivore or the other, as well as a single QTL that controls resistance to both insects. Comparison of QTL for herbivory, glucosinolates, and myrosinase showed that T. ni herbivory is strongly deterred by higher glucosinolate levels, faster breakdown rates, and specific chemical structures. In contrast, P. xylostella herbivory is uncorrelated with variation in the glucosinolate-myrosinase system. This agrees with evolutionary theory stating that specialist insects may overcome host plant chemical defenses, whereas generalists will be sensitive to these same defenses.     

Chronic leptin administration increases serum NEFA in the pig and differentially regulates PPAR expression in adipose tissue

Two in vivo studies were conducted with pigs to determine the effects of exogenous leptin on the expression of peroxisome proliferator activated receptors (PPAR), and on serum concentrations of selected metabolites and hormones. Initially, leptin was administered i.m. to young pigs for 15 days at 0 (control), 0.003 (low), 0.01 (medium) and 0.03 (high) mg. kg(-1). day(-1). There was no leptin effect on serum glucose (P > 0.84), triglycerides (P > 0.69), non-esterified fatty acids (NEFA, P > 0.53), or glycerol (P > 0.33). Leptin at the intermediate and high doses depressed adipose expression of both PPARgamma1 (P < 0.06) and PPARgamma2 (P < 0.01). In a second study, we used a paired-feeding experimental design to determine the effects of a higher dose of leptin (0.05 mg. kg(-1). day(-1)) on serum metabolites and PPAR expression in selected tissues. At this dose, leptin increased (P < 0.0001) serum NEFA concentrations relative to both the ad libitum and pair-fed control groups. However, in this study, there was no difference in the expression of PPARgamma1 in adipose tissue, but PPARgamma2 mRNA was upregulated by leptin (P < 0.08). In contrast, leptin had no impact on the expression of PPARalpha in liver, skeletal muscle or adipose tissue. Adipose tissue explants were also incubated with leptin to assess the effect on PPARgamma expression, in vitro. The abundance of PPARgamma1 mRNA (P < 0.05) was increased after 24 hr of exposure, but the effect of leptin on gamma2 was not significant (P > 0.24). The lipolytic effect of leptin was also evaluated in vitro using isolated adipocytes. In keeping with the increase in serum NEFA concentrations in vivo, leptin stimulated lipolysis in vitro, increasing glycerol concentrations in the medium to about 219% of that in basal (non-treated) culture medium after 8 hr of incubation. Collectively, the data presented herein indicate that leptin modulates lipid metabolism in the pig, but that PPARalpha expression is not a parallel target of leptin as it is in rodent models. The regulation of PPARgamma by leptin seems complex in that it varied in relation to dose in vivo, and may be impacted by in vitro vs. in vivo circumstances.     

Laser Speckle Imaging: A Novel Method for Detecting Dental Erosion

Erosion is a highly prevalent condition known as a non-carious lesion that causes progressive tooth wear due to chemical processes that do not involve the action of bacteria. Speckle images proved sensitive to even minimal mineral loss from the enamel. The aim of the present study was to investigate the use of laser speckle imaging analysis in the spatial domain to quantify shifts in the microstructure of the tooth surface in an erosion model. 32 fragments of the vestibular surface of bovine incisors were divided in for groups (10 min, 20 min. 30 min and 40 min of acid etching) immersed in a cola-based beverage (pH approximately 2.5) twice a day during 7 days to create an artificial erosion. By analyzing the laser speckle contrast map (LASCA) in the eroded region compared to the sound it was observed that the LASCA map shifts, proportionally to the acid each duration, by: 18%; 23%; 39% and 44% for the 10 min; 20 min; 30 min and 40 min groups, respectively. To the best of our knowledge, this is the first study to demonstrate the correlation between speckle patterns and erosion progression.     

Use of transposon promoter-probe vectors in the metabolic engineering of the obligate methanotroph Methylomonas sp. strain 16a for enhanced C40 carotenoid synthesis

The recent expansion of genetic and genomic tools for metabolic engineering has accelerated the development of microorganisms for the industrial production of desired compounds. We have used transposable elements to identify chromosomal locations in the obligate methanotroph Methylomonas sp. strain 16a that support high-level expression of genes involved in the synthesis of the C(40) carotenoids canthaxanthin and astaxanthin. with three promoterless carotenoid transposons, five chromosomal locations-the fliCS, hsdM, ccp-3, cysH, and nirS regions-were identified. Total carotenoid synthesis increased 10- to 20-fold when the carotenoid gene clusters were inserted at these chromosomal locations compared to when the same carotenoid gene clusters were integrated at neutral locations under the control of the promoter for the gene conferring resistance to chloramphenicol. A chromosomal integration system based on sucrose lethality was used to make targeted gene deletions or site-specific integration of the carotenoid gene cluster into the Methylomonas genome without leaving genetic scars in the chromosome from the antibiotic resistance genes that are present on the integration vector. The genetic approaches described in this work demonstrate how metabolic engineering of microorganisms, including the less-studied environmental isolates, can be greatly enhanced by identifying integration sites within the chromosome of the host that permit optimal expression of the target genes.     

Experimental and probabilistic analysis of distal femoral periprosthetic fracture: a comparison of locking plate and intramedullary nail fixation. Part B: probabilistic investigation

The following is Part B of a two-part study. Part A evaluated, biomechanically, intramedullary (IM) nails versus locking plates for fixation of an extra-articular, metaphyseal wedge fracture in synthetic osteoporotic bone. Part B of this study introduces deterministic finite element (FE) models of each construct type in synthetic osteoporotic bone and investigates the probability of periprosthetic fracture of the locking plate compared with the retrograde IM nail using Monte Carlo simulation. Deterministic FE models of the fractured femur implanted with IM nail and locking plate, respectively, were developed and validated using experimental data presented in Part A of this study. The models were validated by comparing the load-displacement curve of the experimental data with the load-displacement curve of the FE simulation with a root-mean square error of less than 3?mm. The validated FE models were then modified by defining the cortical and cancellous bone modulus of elasticity as uncertain variables that could be assumed to vary randomly. Monte Carlo simulation was used to evaluate the probability of fracture (POF) of each fixation. The POF represents the cumulative probability that the predicted shear stresses in the cortical bone will exceed the expected shear strength of the cortical bone. This investigation provides information regarding the significance of post-operative damage accumulation on the POF of the implanted bones when the two fixations are used. The probabilistic analysis found the locking plate fixation to have a higher POF than the IM nail fixation under the applied loading conditions (locking plate 21.8% versus IM nail 0.019%).