Localization of99mTc in bone by means of autoradiography

The uptake of two different preparations of^99mTechnetium-methylene diphosphonate in fetal rat calvaria is compared. The localization of^99mTc after administration of^99mTc(Sn)-MDP and^99mTc-MDP showed equal distribution in autoradiography.     

A comparison of the changes in the non‐neuronal cell populations of the superior cervical ganglia following decentralization and axotomy

Transecting the axons of neurons in the adult superior cervical ganglion (SCG; axotomy) results in the survival of most postganglionic neurons, the influx of circulating monocytes, proliferation of satellite cells, and changes in neuronal gene expression. In contrast, transecting the afferent input to the SCG (decentralization) results in nerve terminal degeneration and elicits a different pattern of gene expression. We examined the effects of decentralization on macrophages in the SCG and compared the results to those previously obtained after axotomy. Monoclonal antibodies were used to identify infiltrating (ED1+) and resident (ED2+) macrophages, as well as macrophages expressing MHC class II molecules (OX6+). Normal ganglia contained ED2+ cells and OX6+ cells, but few infiltrating macrophages. After decentralization, the number of infiltrating ED1+ cells increased in the SCG to a density about twofold greater than that previously seen after axotomy. Both the densities of ED2+ and OX6+ cells were essentially unchanged after decentralization, though a large increase in OX6+ cells occurred after axotomy. Proliferation among the ganglion's total non‐neuronal cell population was examined and found to increase about twofold after decentralization and about fourfold after axotomy. Double‐labeling experiments indicated that some of these proliferating cells were macrophages. After both surgical procedures, the percentage of proliferating ED2+ macrophages increased, while neither procedure altered the proliferation of ED1+ macrophages. Axotomy, though not decentralization, increased the proliferation of OX6+ cells. Future studies must address what role(s) infiltrating and/or resident macrophages play in regions of decentralized and axotomized neurons and, if both are involved, whether they play distinct roles. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 68–79, 2002     

Analysis of folate cofactor levels in tissues using high-performance liquid chromatography

A method for the isolation and concentration of the monoglutamate forms of folate cofactors from tissues and for their subsequent separation and quantitation using HPLC coupled with uv detection at 284 nm is described. A chromatographic procedure utilizing Dowex 50 has been developed for the separation of the folate monoglutamates from a large portion of the nonfolate-related material following digestion of the polyglutamated froms with a highly purified preparation of rat liver conjugase. This chromatographic procedure combined with concentration of the Dowex eluate by lyophilization eliminates uv-absorbing material, which interferes with the detection and quantitation of the folate cofactors and makes possible uv measurement of the individual folates. Reverse-phase paired-ion chromatography on μBondapak C₁₈ coupled with uv detection allows direct quantitation of the folates in the nanogram range.     

Interleukin-1-β and Tumor Necrosis Factor-α Increase Peripheral-Type Benzodiazepine Binding Sites in Cultured Polygonal Astrocytes

Peripheral-type benzodiazepine binding sites (PTBBS) are markedly increased in the injured CNS. Astrocytes appear to be the primary cell type which express increased PTBBS. Because certain cytokines within the injured CNS are potent mitogens for astrocytes, we examined the effects of two such cytokines, interleukin (IL)-1 beta and tumor necrosis factor (TNF), on PTBBS in cultured astrocytes using [3H]Ro 5-4864 as the specific ligand. Purified cultures of either polygonal or process-bearing astrocytes were prepared from neonatal rat cerebral hemispheres. At a concentration of 1.8 nM, specific binding of the radioactive ligand to polygonal astrocytes reached equilibrium within 60 min and was half-maximal by 5-10 min. By contrast, specific binding to process-bearing astrocytes barely exceeded background levels. IL-1 and TNF increased PTBBS within polygonal astrocytes in both dose- and time-dependent manners. At 10-50 ng/ml, IL-1 beta and TNF-alpha elevated [3H]Ro 5-4864 binding in polygonal astrocyte cultures 65 and 87%, respectively, above the level in control cultures. However, no changes in PTBBS were seen within polygonal astrocytes after IL-2 treatment. Scatchard analysis of saturation binding experiments suggested that the increase in PTBBS promoted by TNF was due to an increased number of binding sites present in polygonal astrocytes and not due to an increase in receptor affinity. Binding data suggested that PTBBS within cultures of process-bearing astrocytes were virtually absent irrespective of the treatment. These in vitro data suggest that certain cytokines found in the injured brain may be involved in up-regulating PTBBS within a particular subtype of astrocyte.     

Advances in alternative DNA delivery techniques

This review describes recent advances in alternative DNA-delivery techniques with particular emphasis on silicon carbide fibers, intact tissue electroporation, electrophoresis and microinjection. The advantages/disadvantages of each method along with a historical overview and theory of practice are discussed.     

Dual role of osteoblastic proenkephalin derived peptides in skeletal tissues

Proenkephalin encodes a group of small peptides with opiate-like activity, the endogenous opioids, known to function as neurohormones, neuromodulators, and neurotransmitters. Recently, we have demonstrated that in addition to its abundance in fetal brain tissue, proenkephalin is highly expressed in nondifferentiated mesodermal cells of developing fetuses. We identified the skeletal tissues, bone, and cartilage as major sites of proenkephalin expression. To examine the possibility that proenkephalin is involved in bone development we have studied the expression of this gene in bone-derived cells, its modulation by bone active hormones, and the effects of enkephalin-derived peptides on osteoblastic phenotype. Our studies revealed that osteoblastic cells synthesize high levels of proenkephalin mRNA which are translated, and the derived peptides are secreted. Reciprocal interrelationships between osteoblast maturation and proenkephalin expression were established. These results together with our observations demonstrating inhibitory effects of proenkephalin-derived peptides on osteoblastic alkaline phosphatase activity, strongly support the notion that proenkephalin is involved in bone development. A different direction of research by other investigators has established the capability of the opioid system in the periphery to participate in the control of pain. On the basis of these two lines of observation, we would like to present the following hypothesis: The potential of embryonic skeletal tissue to synthesize proenkephalin-derived peptides is retained in the adult in small defined undifferentiated cell populations. This potential is realized in certain situations requiring rapid growth, such as remodeling or fracture repair. We suggest that in these processes, similarly to the situation in the embryo, the undifferentiated dividing cells produce the endogenous opioids. In the adult these peptides may have a dual function, namely participating in the control of tissue regeneration and in the control of pain. © 1994 Wiley-Liss, Inc.     

The effects of culture medium rigidity on adventitious bud production and tissue vitrification in needle cultures of Sitka spruce [Picea sitchensis (Bong.) Carr.]

Adventitious bud formation on Sitka spruce [ Picca sitchensis (Bong.) Carr.] needle explants was strongly dependent upon the rigidity of the culture medium. In general, of organogenesis was greatest on weak gels and poorest on more rigid gels resulting from increased medium pH or agar strength. There was a significant interaction between agar strength and pH, with the optimum pH for organogenesis declining with increasing agar strength. Poor organogenesis at high agar concentrations was not due to toxic impurities since increased adventitious bud production could be stimulated by decreasing the medium pH whilst maintaining a high agar strength and an agar washing treatment had no significant effect. Although high levels of organogenesis could be sustained on weak gels the resultant adventitious shoots often showed severe vitrification. The frequency of shoots showing vitrification could be reduced by growing the tissues on harder media but this resulted in reduced shoot elongation. Vitrification of needle tissues did not stimulate the formation of adventitious buds in the absence of cytokinins.     

Frost Injury as a Possible Inciting Factor in Bud and Shoot Necroses of Fraxinus excelsior L.

Large numbers of European ash have died in Poland in all age classes during the last ten years. The characteristic symptom occurring on shoots of planted and self‐sown seedlings was bark necroses starting from the shoot apex, necrotic buds, or leaf and twig scars. The results showed that in the bud tissue of cold acclimated European ash extracellular and intracellular ice formation occurred at approximately ?9 and ?32°C, respectively. In deacclimated plants in spring water supercooling is limited by the heterogenous ice nucleation temperature and consequently the cold tolerance is ?9 to ?4°C for bud tissues and ?13 to ?9°C for shoots. Isolations of fungi were performed from dead buds and from necroses occurring on the main stem. Alternaria alternata, Fusarium lateritium and Phomopsis scobina were among the fungi occurring in both these organs at frequencies of more than 7%. Cylindrocarpon heteronemum, Diplodia mutila and Tubercularia vulgaris from necroses were only isolated in frequencies; 3.3, 1.2 and 5.4%, respectively. It seems likely that freezing injury is the inciting factor, which combined with fungal colonization manifests itself as fatal damage to European ash buds and shoots.     

High-dose mode of death in Tribolium

This study reports the first demonstration within a single insect genus (Tribolium) of both the acute, or lethal-midlethal, dose-independent pattern of mortality, and the hyperacute, dose-dependent pattern, after appropriate doses of ionizing radiation. This demonstration provides resolution of apparently contradictory reports of insect responses in terms of doses required to cause lethality and those based on survival time as a function of dose. A dose-dependent mortality pattern was elicited in adult Tribolium receiving high doses, viz., 300 Gy or greater; its time-course was complete in 10 days, before the dose-independent mortality began. Visual observations of heavily-irradiated Tribolium suggested neural and/or neuromuscular damage, as had been previously proposed by others for lethally-irradiated wasps, flies, and mosquitoes. Results of experiments using fractionated high doses supported the suggestion that the hyperacute or high-dose mode of death is the result of damage to nonproliferative tissues. Relative resistance of a strain to the hyperacute or high-dose mode of death is not necessarily correlated with resistance to the midlethal mode, which is believed to be the result of damage to the proliferative cells of the midgut.

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Résumé Les résultats de nombreuses études des réactions des insectes adultes de différents groupes à l'irradiation s'opposent quant à l'importance de la dose provoquant la létalité et quant aux modalités de la mort. Les diptères et les guèpes impliquent des doses très élevées,-des centaines de Gy-, ne présentent aucune période caractérisant la mort par irradiation, et décèdent de plus en plus tôt avec l'augmentation des doses. Beaucoup d'autres insectes succombent à des doses (milétalelétale) beaucoup plus faibles,-de quelques Gy à des dizaines-, et quelle que soit la dose meurent au bout d'un temps voisin.Au cours de cette étude, nous avons pu observer que ces deux types de mortalité peuvent être provoqués chez le même genre d'insecte (Tribolium), avec des doses convenables d'irradiation . Un syndrôme caractéristique a été provoqué avec des doses très élevées, de 300 Gy ou plus,-à ces doses la mort est obtenue en 10 jours après l'irradiation. L'absence de syndrôme caractéristique se produit avec des doses inférieures ou égales à 80 Gy; la mort a lieu alors entre 10 et 16 jours en fonction de la dose.Les différences entre les deux types de décès indiquent deux processus de mort par irradiation. La manifestation d'une désorientation et d'une perte de coordination motrice chez les Tribolium fortement irradiés suggère des altérations neurales et/ou neuromusculaires comme cause/s de ce type de mort provoquée par des doses élevées. L'implication de tissus sans prolifération a été confortée par les résultats d'expériences utilisant de hautes doses fractionnées. Le type de mort milétal est considéré après de nombreuses observations indépendantes, comme le résultat d'atteintes à la prolifération des cellules de l'intestin moyen.Les données contradictoires sur les réactions des insectes aux irradiations proviennent d'abord de l'absence de connaissances sur le lieu des dégâts. Les diptères sont connus maintenant, après différentes études, comme perdant complètement l'aptitude au renouvellement cellulaire, et présentent ainsi un type de mort avec dose élevée. Beaucoup d'autres insectes adultes ont un renouvellement cellulaire de l'intestin moyen limité, et ainsi présentent le type de mort milétal. Le type de mort, dit haute dose, peut être induit dans cette dernière catégorie d'insectes par une irradiation suffisamment forte, et, dans le cas du Tribolium le déroulement de la mort se produit alors de deux façons bien distinctes.