Na+-K+-ATPase in frog esophagus mucociliary cell membranes: inhibition by protein kinase C activation

We examined protein kinase C (PKC)-dependentregulation ofNa⁺-K⁺-ATPasein frog mucociliary cells. Activation of PKC by12-O-tetradecanoylphorbol-13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol(diC8) either in intact cells or isolated membranes resulted in aspecific inhibition ofNa⁺-K⁺-ATPaseactivity by ~25-45%. The inhibitory effects in membranes exhibited time dependence and dose dependence [half-maximalinhibition concentration (IC₅₀) = 0.5 ± 0.1 nM and 2.4 ± 0.2 µM, respectively, for TPA anddiC8] and were not influenced byCa²⁺. Analysis of the ouabaininhibition pattern revealed the presence of twoNa⁺-K⁺-ATPaseisoforms with IC₅₀ values forcardiac glycoside of 2.6 ± 0.8 nM and 409 ± 65 nM,respectively. Most importantly, the isoform possessing a higheraffinity for ouabain was almost completely inhibited by TPA, whereasits counterpart was hardly sensitive to the PKC activator. The resultssuggest that, in frog mucociliary cells, PKC regulatesNa⁺-K⁺-ATPaseand that this action is related to the specificNa⁺-K⁺-ATPaseisoform.

     

EFFECT OF NITROGEN STARVATION ON OPTICAL PROPERTIES,PIGMENTS, AND ARACHIDONIC ACID CONTENT OF THE UNICELLULAR GREEN ALGA PARIETOCHLORIS INCISA (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

Spectral properties of cell suspensions, individual cells, and extracts of the unicellular green alga Parietochloris incisa (Reisigl) Shin Watan. grown under low light were studied. Long‐term nitrogen (N) deprivation resulted in a decrease of chloroplast volume, appearance of numerous large cytoplasmic oil bodies, and the deposition of triacylglycerols with a high proportion of arachidonic acid. Chlorophylls a and b underwent a synchronous decline, whereas carotenoids (Car) showed a relative increase. Simultaneously, significant qualitative changes in the spectral properties of P. incisa individual cells, cell extracts, and cell suspensions were observed. To a large extent, the spectral changes observed in cell suspension could be attributed to a decrease in overall pigment content, leading to a gradual weakening of the so‐called package effect and accumulation of additional amounts of Car over chl, most probably, in oil bodies. Several optical characteristics of cell suspensions could serve as sensitive indicators of N‐deficiency in P. incisa. Furthermore, the absorption ratios, A₄₇₆/A₆₇₆ and A₆₅₀/A_676, showed close correlations with the Car‐to‐chl ratio and relative arachidonic acid (AA) content, respectively. The latter makes it possible to suggest that the increase in AA percentage in P. incisa proceeds in parallel with a decrease in cell chl content, accounting for the weakening of the package effect. N‐replenishment resulted in complete recovery of cell optical properties. The possible significance of the changes in cell ultrastructure, pigments, lipids, and optical properties is discussed with special reference to the ability of algae to adapt to and survive under conditions of long‐term nutrient deficiency.     

In vivo evidence for translesion synthesis by the replicative DNA polymerase δ

The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro. These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass.     

Forces applied by cilia measured on explants from mucociliary tissue

Forces applied by intact mucus-propelling cilia were measured for the first time that we know of using a combined atomic force microscopy (AFM) and electrooptic system. The AFM probe was dipped into a field of beating cilia and its time-dependent deflection was recorded as it was struck by the cilia while the electrooptic system simultaneously and colocally measured the frequency to ensure that no perturbation was induced by the AFM probe. Using cilia from frog esophagus, we measured forces of ~0.21 nN per cilium during the effective stroke. This value, together with the known internal structure of these cilia, leads to the conclusion that most dynein arms along the length of the axoneme contribute to the effective stroke of these cilia.     

Direct involvement of p53 in the base excision repair pathway of the DNA repair machinery

The p53 tumor suppressor that plays a central role in the cellular response to genotoxic stress was suggested to be associated with the DNA repair machinery which mostly involves nucleotide excision repair (NER). In the present study we show for the first time that p53 is also directly involved in base excision repair (BER). These experiments were performed with p53 temperature-sensitive (ts) mutants that were previously studied in in vivo experimental models. We report here that p53 ts mutants can also acquire wild-type activity under in vitro conditions. Using ts mutants of murine and human origin, it was observed that cell extracts overexpressing p53 exhibited an augmented BER activity measured in an in vitro assay. Depletion of p53 from the nuclear extracts abolished this enhanced activity. Together, this suggests that p53 is involved in more than one DNA repair pathway.     

Insulin--a growth hormone

Insulin is a peptide hormone with a high degree of homology with the insulin-like growth factor I (IGF-I). Its biological actions are interlaced with those of GH and IGF-I. The objective of this study is to review the growth promoting actions of insulin. The experimental evidence consists of the use of organ cultures of neonatal mice condilar cartilage insulin which stimulates the cartilage cell differentiation and maturation. Injection of insulin to hypohysectomized rats stimulated tibial growth. Clinical evidence is manifold. Babies with diabetes and hypoinsulinemia are short, whereas babies with hyperinsulinism are big. Children with idiopathic short stature have low insulin whereas obese children with hyperinsulinism are tall. Hypo-insulinized children with diabetes slow their growth until the insulin dose is optimized. It remains to be clarified whether insulin exerts its growth promoting actions via its own receptors, via the IGF-I receptors, or via a hybrid (insulin--IGF-I) receptor.     

Chronic parasite infections cause immune changes that could affect successful vaccination

Several important issues must be considered when performing any vaccination in areas with high prevalence of geohelminths. Immunization of populations infected with geohelminths could be sub-optimal if the immune background is not taken into consideration. Immune modulation and deworming might be essential for effective protective vaccination. In addition, further animal models and clinical studies addressing these issues are required. Underscoring the importance of these issues, a recent report has highlighted several vaccination studies in which nematode-infected mice or pigs failed to mount efficient protective immune responses.     

Grain zinc, iron and protein concentrations and zinc-efficiency in wild emmer wheat under contrasting irrigation regimes

Micronutrient malnutrition, and particularly deficiency in zinc (Zn) and iron (Fe), afflicts over three billion people worldwide, and nearly half of the world’s cereal-growing area is affected by soil Zn deficiency. Wild emmer wheat [Triticum turgidum ssp. dicoccoides (Körn.) Thell.], the progenitor of domesticated durum wheat and bread wheat, offers a valuable source of economically important genetic diversity including grain mineral concentrations. Twenty two wild emmer wheat accessions, representing a wide range of drought resistance capacity, as well as two durum wheat cultivars were examined under two contrasting irrigation regimes (well-watered control and water-limited), for grain yield, total biomass production and grain Zn, Fe and protein concentrations. The wild emmer accessions exhibited high genetic diversity for yield and grain Zn, Fe and protein concentrations under both irrigation regimes, with a considerable potential for improvement of the cultivated wheat. Grain Zn, Fe and protein concentrations were positively correlated with one another. Although irrigation regime significantly affected ranking of genotypes, a few wild emmer accessions were identified for their advantage over durum wheat, having consistently higher grain Zn (e.g., 125 mg kg^?1), Fe (85 mg kg^?1) and protein (250 g kg^?1) concentrations and high yield capacity. Plants grown from seeds originated from both irrigation regimes were also examined for Zn efficiency (Zn deficiency tolerance) on a Zn-deficient calcareous soil. Zinc efficiency, expressed as the ratio of shoot dry matter production under Zn deficiency to Zn fertilization, showed large genetic variation among the genotypes tested. The source of seeds from maternal plants grown under both irrigation regimes had very little effect on Zn efficiency. Several wild emmer accessions revealed combination of high Zn efficiency and drought stress resistance. The results indicate high genetic potential of wild emmer wheat to improve grain Zn, Fe and protein concentrations, Zn deficiency tolerance and drought resistance in cultivated wheat.