Nerve Growth Factor Stimulates Multisite Tyrosine Phosphorylation and Activation of the Atypical Protein Kinase C's via a src Kinase Pathway

Atypical protein kinase C (PKC) isoforms are required for nerve growth factor (NGF)-initiated differentiation of PC12 cells. In the present study, we report that PKC-iota becomes tyrosine phosphorylated in the membrane coincident with activation posttreatment with nerve growth factor. Tyrosine phosphorylation and activation of PKC-iota were inhibited in a dose-dependent manner by both PP2 and K252a, src and TrkA kinase inhibitors. Purified src was observed to phosphorylate and activate PKC-iota in vitro. In PC12 cells deficient in src kinase activity, both NGF-induced tyrosine phosphorylation and activation of PKC-iota were also diminished. Furthermore, we demonstrate activation of src by NGF along with formation of a signal complex including the TrkA receptor, src, and PKC-iota. Recruitment of PKC-iota into the complex was dependent on the tyrosine phosphorylation state of PKC-iota. The association of src and PKC-iota was constitutive but was enhanced by NGF treatment, with the src homology 3 domain interacting with a PXXP sequence within the regulatory domain of PKC-iota (amino acids 98 to 114). Altogether, these findings support a role for src in regulation of PKC-iota. Tyrosine 256, 271, and 325 were identified as major sites phosphorylated by src in the catalytic domain. Y256F and Y271F mutations did not alter src-induced activation of PKC-iota, whereas the Y325F mutation significantly reduced src-induced activation of PKC-iota. The functional relevance of these mutations was tested by determining the ability of each mutant to support TRAF6 activation of NF-kappaB, with significant impairment by the Y325F PKC-iota mutant. Moreover, when the Y352F mutant was expressed in PC12 cells, NGF's ability to promote survival in serum-free media was reduced. In summary, we have identified a novel mechanism for NGF-induced activation of atypical PKC involving tyrosine phosphorylation by c-Src.     

Observations on Alexandrium tamarense (Lebour) Balech and Other Dinoflagellate Populations in Golfo Nuevo, Patagonia (Argentina)

The toxic dinoflagellate Alexandrium tamarense and other dinoflagellatespecies were studied, along with water temperature and nutrientconcentrations, from September 1995 to December 1998 in theGolfo Nuevo, Chubut, Argentina. Nutrient concentrations werelow, showing a peak of high concentration in winter and a phaseof depletion in late spring and summer. Dinoflagellates tendedto be abundant during spring and summer, when Prorocentrum micanswas the most important species. Other dinoflagellates were Pyrophacushorologium and Dinophysis acuminata. Ceratium tripos, C. fususand C. horridum were present during the autumn, and a C. tripospeak up to 5.9 x 10³ cell l^–1 was observed in May 1997.Alexandrium tamarense showed strong interannual variation, thehighest concentration being found in spring (September–October)1995, with densities up to 15 x 10³ cells l^–1. The secondA.tamarense peak was observed during October–November1998 with maximal densities up to 5 x 10³ cells l^–1. Moderatelyhigh A. tamarense cyst densities, up to 300 cysts cm^–3of sediment, were found in the deep zone of the Golfo Nuevobasin. Among meteorolog-ical variables, increased late winterrain and higher solar radiation during spring may have influencedA. tamarense blooms.     

Virtual brain endocast of Antifer (Mammalia: Cervidae), an extinct large cervid from South America

A diverse fossil record of Cervidae (Mammalia) has been documented in the South American Pleistocene, when these animals arrived during the Great American Biotic Interchange. Using computed tomography-scanning techniques, it is possible to access the endocranial morphology of extinct species. Here, we studied the brain endocast of the extinct late Pleistocene cervid Antifer ensenadensis from southern Brazil, one of the largest forms that lived on this continent, using comparative morphology, geometric morphometrics, and encephalization quotients. The analyzed endocasts demonstrate that A. ensenadensis had a gyrencephalic brain, showing a prominent longitudinal sinus (=sagittal superior sinus), which is also observed in the large South American cervid Blastocerus dichotomus. The encephalization quotient is within the variation of extant cervids, suggesting maintenance of the pattern of encephalization from at least the late Pleistocene. Geometric morphometric analysis suggested a clear and linear allometric trend between brain endocast size and shape, and highlights A. ensenadensis as an extreme form within the analyzed cervids regarding brain morphology.     

Metal-controlled interdomain cooperativity in parvalbumins

Conformational behavior of five homologous proteins, parvalbumins (PAs) from northern pike (α and β isoforms), Baltic cod, and rat (α and β isoforms), was studied by scanning calorimetry, circular dichroism, and bis-ANS fluorescence. The mechanism of the temperature-induced denaturation of these proteins depends dramatically on both the peculiarities of their amino acid sequences and on their interaction with metal ions. For example, the pike α-PA melting can be described by two successive two-state transitions with mid-temperatures of 90 and 120 °C, suggesting the presence of two thermodynamic domains. The intermediate state populated at the end of the first transition was shown to bind Ca²⁺ ions, and was characterized by the largely preserved secondary structure and increased solvent exposure of hydrophobic groups. Mg²⁺- and Na⁺-loaded forms of pike α-PA demonstrated a single two-state transition. Therefore, the mechanism of the PA thermal denaturation is controlled by metal binding. It ranged from the absence of detectable first-order transition (apo-form of pike PA), to the two-state transition (e.g., Mg²⁺- and Na⁺-loaded forms of pike α-PA), to the more complex mechanisms (Ca²⁺-loaded PAs) involving at least one partially folded intermediate. Analysis of isolated cavities in the protein structures revealed that the interface between the CD and EF subdomains of Ca²⁺-loaded pike α-PA is much more loosely packed compared with PAs manifesting single heat-sorption peak. The impairment of interactions between CD and EF subdomains may cause a loss of structural cooperativity and appearance of two separate thermodynamic domains. One more peculiar feature of pike α-PA is that depending on its interactions with metal ions, it can be an intrinsically disordered protein (apo-form), an ordered protein of mesophilic (Na⁺-bound state), thermophilic (Mg²⁺-form), or even of the hyperthermophilic origin (Ca²⁺-form).     

Synergistic anti-proliferative effects of vitamin D derivatives and 9-cis retinoic acid in SH-SY5Y human neuroblastoma cells.

This study examines the effect of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)₂D₃ or its derivatives, but significantly decreased in the presence of the two retinoids (0.001–10 μM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060, and 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.     

Effect of lactoferrin on oxidative features of ceruloplasmin

In our previous report we first described a complex between lactoferrin (Lf) and ceruloplasmin (Cp) with K _d ~ 1.8 μM. The presence of this complex in colostrum that never contains more than 0.3 μM Cp questions the reliability of K _d value. We carefully studied Lf binding to Cp and investigated the enzymatic activity of the latter in the presence of Lf, which allowed obtaining a new value for K _d of Cp–Lf complex. Lf interacting with Cp changes its oxidizing activity with various substrates, such as Fe²⁺, o-dianisidine (o-DA), p-phenylenediamine (p-PD) and dihydroxyphenylalanine (DOPA). The presence of at least two binding sites for Lf in Cp molecule is deduced from comparison of substrates’ oxidation kinetics with and without Lf. When Lf binds to the first site affinity of Cp to Fe²⁺ and to o-DA increases, but it decreases towards DOPA and remains unchanged towards p-PD. Oxidation rate of Fe²⁺ grows, while that of o-DA, p-PD and DOPA goes down. Subsequent Lf binding to the second center has no effect on iron oxidation, hampers DOPA and o-DA oxidation, and reduces affinity towards p-PD. Scatchard plot for Lf sorbing to Cp-Sepharose allowed estimating K _d for Lf binding to high-affinity (~13.4 nM) and low-affinity (~211 nM) sites. The observed effect of Lf on ferroxidase activity of Cp is likely to have physiological implications.     

Technical viability of the production,partial purification and characterisation of inulinase using pretreated agroindustrial residues

The present work aimed to study the viability of the use of sugarcane molasses and corn steep liquor (CSL) in a sequential inulinase production performing an up-stream pretreatment of these agroindustrial residues. A sequential strategy was used applying three central composite rotatable designs (CCRDs) to optimise medium composition, followed by a down-stream step. The medium containing 150 g L⁻¹ molasses, 50 g L⁻¹ CSL and 6 g L⁻¹ yeast extract, yielded a maximum inulinase production of 1,294 ± 7 U mL⁻¹, after 72 h of fermentation. A down-stream evaluation was carried out using an expanded bed of Streamline DAE resin (Pharmacia), with and without the up-stream treatment. The results showed that the enzyme could not be recovered from the non-pretreated medium, whereas a yield of 91% was obtained in the adsorption stage from the medium prepared with the up-stream treatment, showing the viability of producing the enzyme inulinase from agroindustrial residues using the integrated process.     

Biosynthesis of fatty acids and triacylglycerols by 2,6,10,14-tetramethyl pentadecane-grown cells of Nocardia globerula 432

Nocardia globerula strain 432 was able to synthesize triacylglycerols (TAG) during cultivation on 2,6,10,14-tetramethyl pentadecane (pristane) under nitrogen-limiting conditions. Within these cells, 4,8,12-trimethyl tridecanoic acid was the major fatty acid detected. Fatty acids with an odd number of carbon atoms and minor amounts of even-numbered fatty acids were also observed. Experiments carried out with acrylic acid, an inhibitor of beta-oxidation, suggested that odd-numbered fatty acids such as C15:0, C17:0 and 10-methyl C17:0 were synthesized de novo using propionyl-CoA, the beta-oxidation product, as precursor. Although N. globerula 432 incorporated mainly straight chain fatty acids into TAG, the branched fatty acid 4,8,12-trimethyl tridecanoic acid also appeared, to some extent, in the acylglycerols. The importance of TAG biosynthesis by pristane-grown cells of N. globerula strain 432 is discussed.     

Phloem‐specific resistance in Brassica oleracea against the whitefly Aleyrodes proletella

The cabbage whitefly [Aleyrodes proletella L. (Hemiptera: Aleyrodidae)] is becoming a serious pest in Brassica oleracea L. (Brassicaceae) crops. However, almost nothing is known about the interaction of this insect with its host plants. Previous studies have shown differences in the natural occurrence of adults, eggs, and nymphs on the closely related B. oleracea cultivars Christmas Drumhead and Riviera grown in the field. In this study, we aimed to identify the nature of these differences and to gain insight into the resistance mechanisms against A. proletella. We used no‐choice experiments on field‐ and greenhouse‐grown plants to show that the differences between the two cultivars are mainly based on antibiosis (traits that reduce herbivore performance) and not on antixenosis (traits that deter herbivory). This was further supported by laboratory choice experiments that indicated little or no discrimination between the two cultivars based on plant volatiles. We showed that resistance is dependent on plant age, that is, resistance increased during plant development, and is mainly independent of environmental factors. Analysis of probing behaviour revealed that the resistance trait affects A. proletella at the phloem level and that morphological differences between the two cultivars are most likely not involved. We suggest that compounds present in the phloem reduce sap ingestion by the whitefly and that this explains the observed resistance.     

Listeria monocytogenes Behaviour in Presence of Non-UV-Irradiated Titanium Dioxide Nanoparticles

Listeria monocytogenes is the agent of listeriosis, a food-borne disease. It represents a serious problem for the food industry because of its environmental persistence mainly due to its ability to form biofilm on a variety of surfaces. Microrganisms attached on the surfaces are a potential source of contamination for environment and animals and humans. Titanium dioxide nanoparticles (TiO₂ NPs) are used in food industry in a variety of products and it was reported that daily exposure to these nanomaterials is very high. Anti-listerial activity of TiO₂ NPs was investigated only with UV-irradiated nanomaterials, based on generation of reactive oxigen species (ROS) with antibacterial effect after UV exposure. Since both Listeria monocytogenes and TiO₂ NPs are veicolated with foods, this study explores the interaction between Listeria monocytogenes and non UV-irradiated TiO₂ NPs, with special focus on biofilm formation and intestinal cell interaction. Scanning electron microscopy and quantitative measurements of biofilm mass indicate that NPs influence both production and structural architecture of listerial biofilm. Moreover, TiO₂ NPs show to interfere with bacterial interaction to intestinal cells. Increased biofilm production due to TiO₂ NPs exposure may favour bacterial survival in environment and its transmission to animal and human hosts.