Radiation response characteristics of human cells in vitro

Improvements in tissue culture techniques and growth media have made it possible to culture a range of cells of human origin, both normal and malignant. The most recent addition to the list are endothelial cells from umbilical cord veins. Interesting results in radiosensitivity studies of these human cells have been obtained, some of which may have implications in radiation therapy. (i) Repair of potentially lethal damage (PLDR) has been observed in all cell lines investigated; cells of normal origin repair PLD at least as well as malignant cells, which makes clinical trials of PLDR inhibitors of doubtful usefulness. (ii) No apparent correlation can be made between the extent of PLDR and the traditional radioresponsiveness of a particular tumor type. Indeed, if anything, it could appear to have an inverse correlation since the most resistant tumor cells show the smallest amount of PLD repair. (iii) Dose-rate effects appear to be better predictors of radiosensitivity than PLDR capacity. (iv) Sublethal damage repair, manifest by a dose-rate effect, has also been observed in all human cell lines tested. Cells of normal tissue origin, including fibroblasts and endothelial cells, exhibit a dose-rate effect that is intermediate between that for cells from traditionally resistant tumors (melanoma and osteosarcoma) and cells from more sensitive tumors (neuroblastoma and breast).     

Population dynamics of Narapa bonettoi Righi and Varela, 1983 (Oligochaeta: Narapidae) from the main channel of the Middle Paraná River,Argentina

The aim of the present study was to determine the life-cycle of Narapa bonettoi in the main channel of the Middle Paraná River. The average density varied between 17 and 624 ind. sample^–1. The spatial distribution was contagious with a higher degree of aggregation during the first months of the observed cycle, than during the last months. Mature specimens were more abundant in September 1985–86, resulting in greater breeding activity in early summer, when there was a large population increase. Two reproductive strategies were observed: asexual and sexual. After breeding, worms undergo a resorption process of their reproductive organs, returning to a non-sexual condition. Narapa bonettoi probably uses asexual reproduction as an adaptative strategy, because cocoon development will be affected by environmental factors such as the drift in the Paraná River which is highly erodable. Population growth was rapid during the first four months and then stabilized.     

Fertility differences among floral morphs following selfing in tristylous Eichhornia paniculata (Pontederiaceae): inbreeding depression or partial incompatibility?

Reduction in seed set following self- vs. cross-pollination in flowering plants can result from abortion of selfed offspring owing to inbreeding depression and/or partial self-incompatibility. Previous studies on tristylous Eichhornia paniculata (Pontederiaceae) indicate that reduced seed set following self-pollination generally occurs in the short- (S), but not the long-(L) or mid-styled (M) morphs. To determine whether this pattern results from morph-specific differences in inbreeding depression owing to the sheltering of deleterious alleles at the S locus and/or partial self-incompatibility, we conducted controlled hand-pollinations of the floral morphs and measured seed set and levels of seed abortion. There were no significant differences in fertilization success and seed set following self-, illegitimate, and legitimate pollinations in the L and M morphs. In contrast, in the S morph self-, intramorph and intermorph illegitimate pollinations resulted in significant reduction in seed set in comparison with legitimate pollination. This indicates that the reduced seed set observed in self-pollination is the result of partial incompatibility rather than inbreeding depression. Significantly reduced fertilization success and low levels of ovule abortion in illegitimate pollinations of S plants also supported this conclusion. Reduced fertility in the S morph may have implications for the observed loss of this morph from natural populations and the evolutionary breakdown of tristyly.     

Production and stabilization of cellulases fromTrichoderma reesei

Summary Production of exoglucanase and endoglucanase in batch culture ofT. reesei QM 9414 was growth-associated both in lactose and leached beet pulp media. Endoglucanase remained stable whilst the organism was in the stationary phase but exoglucanase activityy decreased sharply at the onset of the stationary phase. Addition of the protease inhibitor phenylmethylsulphonyl fluoride (PMSF) towards the end of growth produced complete protection toward protease inactivation, exoglucanase being stabilized even in prolonged stationary phase. The stability of exoglucanase in the presence or absence of mycelium, with and without PMSF addition, showed that endogenous cell-free and cell-associated proteases weree responsible for exoglucanase inactivation in the stationary phase. Inactivation of exoglucanase depended on the physiological stage of the cultures, the enzyme remaining stable as long as the culture was in an active growth phase.

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Resumen La producción de exoglucanase y endoglucanasa deT. reesei QM 9414 en cultivo por lotes en medios con lactosa y coseta agotada de remolacha como fuentes de carbono, fue asociada al crecimiento. La endoglucanasa permaneció estable en fase estacionaria, pero la actividad de exoglucanase disminuyó drásticamente al entrar el cultivo en dicha fase. La adición del inhibidor de proteasas PMSF hacia el final de la fase de crecimiento provocó la total inactivación de las proteasas producidas, lográndose estabilizar la exoglucanasa aún en prolongada fase estacionaria. Se analizó la estabilidad de exoglucanasa en presencia y ausencia de micelio, con y sin adición de PMSF; los resultados muestran que proteasas endogenas extracelulares y asociadas a la célula son responsables de la inestabilidad de la exoglucanasa en fase estacionaria. La inactivación de la exoglucanasa depende del estado fisiológico del cultivo, ya que la enzima permanece estable mientras el cultivo se encuentra en crecimiento.

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Résumé La production d'exoglucanase et d'endoglucanase dans les cultures deT. reesei QM 9414 en milieu non renouvelé est associée à la croissance tant du lactose que du jus d'extraction de la pulpe de betterave. L'endo-glucanase reste stable tant que le microorganisme est dans sa phase stationnaire de croissance tandis que l'activité exoglucanasique décroit rapidement dès le début de cette phase stationnaire. L'ajour du fluorure de phenylmethylsulfonate (PMSF), un inhibiteur protéolytique, vers la fin de la croissance a exercé une protection complète contre l'inactivation par la protéase: l'exoglucanase était protégée dans ces conditions même en phase stationnaire prolongée. La stabilité de l'exoglucanase en présence ou en absence de mycelium, avec ou sans ajout de PMSF démontre la responsabilité des exo-protéases endogènes solubles et de protéases associées aux cellules dans l'inactivation de l'exoglucanase dans la phase stationnaire. L'inactivation de l'exoglucanase dépend de l'état physiologique des cultures. En effet, l'enzyme reste stable aussi longtemps que la culture est en phase active de croissance.

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Based on a paper presented at the First Latin American Congress on Biotechnology, Tucumán, Argentina, October 4–8, 1987.     

Modifications in DARPP‐32 phosphorylation pattern after repeated palatable food consumption undergo rapid habituation in the nucleus accumbens shell of non‐food‐deprived rats

In non‐food‐deprived rats a palatable meal induces a transient increase in dopamine output in the prefrontal cortex and nucleus accumbens shell and core; habituation to this response develops with a second palatable meal, selectively in the shell, unless animals are food‐deprived. A palatable meal also induces time‐dependent modifications in the dopamine and cAMP‐regulated phosphoprotein of Mr 32 000 (DARPP‐32) phosphorylation pattern that are prevented when SCH 23390, a selective dopamine D₁ receptor antagonist, is administered shortly after the meal. This study investigated whether dopaminergic habituation in the shell had a counterpart in DARPP‐32 phosphorylation changes. In non‐food‐deprived rats, two consecutive palatable meals were followed by similar sequences of modifications in DARPP‐32 phosphorylation levels in the prefrontal cortex and nucleus accumbens core, while changes after the second meal were blunted in the shell. In food‐deprived rats two consecutive meals also induced similar phosphorylation changes in the shell. Finally, SCH 23390 administered shortly after the first palatable meal in non‐food‐deprived rats inhibited DARPP‐32 phosphorylation changes in response to the first meal, and prevented the habituation to a second meal in terms of dopaminergic response and DARPP‐32 phosphorylation changes. Thus, dopamine D₁ receptor stimulation plays a role in the development of habituation.     

A short PNA targeting coxsackievirus B3 5'-nontranslated region prevents virus-induced cytolysis.

Targeting regulatory RNA regions to interfere with the biosynthesis of a protein is an intriguing alternative to targeting a protein itself. Regulatory regions are often unique in sequence and/or structure and, thus, ideally suited for specific recognition with a low risk of undesired side effects. Targeting regulatory RNA elements, however, is complicated by their complex three-dimensional structure, which poses kinetic and thermodynamic constraints to the recognition by a complementary oligonucleotide. Oligonucleotide mimics, which shift the thermodynamic equilibrium towards complex formation and yield stable complexes with a target RNA, can overcome this problem. Peptide nucleic acids (PNA) represent such a promising class of molecules. PNA are very stable, non-ionic compounds and they are not sensitive to enzymatic degradation. Yet, PNA form specific base pairs with a target sequence. We have designed, synthesised and characterised PNA able to enter infected cells and to bind specifically to a control region of the genomic RNA of coxsackievirus B3 (CVB3), which is an important human pathogen. The results obtained by studying the interaction of such PNA with their RNA target, the entrance into the cell and the viral inhibition are herein presented.