Reactive Oxygen Species Produced by a Photodynamic Effect Induced Calcium Signal in Neurons and Astrocytes

Photodynamic therapy (PDT) leads to production of reactive oxygen species (ROS) and cell destruction due to oxidative stress. We used photodynamic effect of photosensitizer radachlorin to unravel the effect of photo-induced oxidative stress on the calcium signal and lipid peroxidation in primary culture of cortical neurons and astrocytes using live cell imaging. We have found that irradiation in presence of 200 nM of radachlorin induces calcium signal in primary neurons and astrocytes. Photo-induced neuronal calcium signal depends on internal calcium stores as it was still observed in calcium-free medium and could be blocked by depletion of endoplasmic reticulum (ER) stores with inhibitor of sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA) thapsigargin. Both inhibitors of phospholipase C activity U73122 and water-soluble analogue of vitamin E Trolox suppressed calcium response activated by PDT. We have also observed that the photodynamic effect of radachlorin induces lipid peroxidation in neurons and astrocytes. This data demonstrate that lipid peroxidation induced by PDT in neurons and astrocytes leads to activation of phospholipase C that results in production of inositol 1,4,5-trisphosphate (IP3).     

Receptor recognition sites of cytokines are organized as exchangeable modules. Transfer of the leukemia inhibitory factor receptor-binding site from ciliary neurotrophic factor to interleukin-6

Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4-helical bundle" cytokines of the IL-6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology.     

A Route to Direct Fitness: Natural and Experimentally Induced Queen Succession in the Tropical Primitively Eusocial Wasp <Emphasis Type="Italic">Ropalidia marginata</Emphasis>

Insect societies are hallmarks of cooperation because one or a few queens monopolize reproduction and several non-reproductive workers cooperatively raise brood. However, the loss of the queen exposes a colony to potential reproductive conflict, which is resolved only after a new queen takes over. We studied queen succession in natural and experimental colonies of the primitively eusocial wasp Ropalidia marginata to understand the proximate behavioral strategies involved in the resolution of this conflict. Previous work has shown that in this species, experimental queen removal always results in only one worker becoming hyper-aggressive and taking over the colony as its next queen, without ever being challenged. Here we show that even during natural queen turnover, one and only one worker becomes hyper-aggressive and takes over as the next queen, without being challenged. During natural queen turn-over, aggression of the successor may sometimes begin before the loss of the old queen and may sometimes decline more rapidly, unlike in the case of experimental queen removal. The successor begins to lay eggs sooner after a natural queen turn-over as compared to experimental queen removal. This is expected because workers might detect the gradual decline of the queen preceding her disappearance. Because queen succession is expected to be more prevalent in tropical perennial species, we expect natural selection to have favored such an orderly queen succession so that a route to direct fitness is available without significant reduction in cooperation.     

Genotyping of Polyomavirus BK by Real Time PCR for VP1 Gene

Polyomavirus BK latently persist in different sites, including the renourinary tract, and may reactivate causing nephropathy in renal transplant recipients or hemorrhagic cystitis in bone marrow recipients. Based on the sequence of the VP1 gene, four genotypes have been described, corresponding to the four serologically differentiated subtypes I–IV, with different prevalence and geographic distribution. In this study, the development and clinical validation of four different Real-Time PCR assays for the detection and discrimination of BKV genotypes as a substitute of DNA sequencing are described. 379 BK VP1 sequences, belonging to the main four genotypes, were aligned and “hot spots” of mutation specific for all the strains or isolates were identified. Specific primers and probes for the detection and discrimination of each genotype by four Real-Time PCR assays were designed and technically validated. Subsequently, the four Real-Time PCR assays were used to test 20 BK-positive urine specimens from renal transplant patients, and evidenced a prevalence of BK genotype I, as previously reported in Europe. Results were confirmed by sequencing. The availability of a rapid and simple genotyping method could be useful for the evaluation of BK genotypes prevalence and studies on the impact of the infecting genotype on viral biological behavior, pathogenic role, and immune evasion strategies.     

Nucleolar retention of a translational C/EBPα isoform stimulates rDNA transcription and cell size

The messenger RNA of the intronless CEBPA gene is translated into distinct protein isoforms through the usage of consecutive translation initiation sites. These translational isoforms have distinct functions in the regulation of differentiation and proliferation due to the presence of different N‐terminal sequences. Here, we describe the function of an N‐terminally extended protein isoform of CCAAT enhancer‐binding protein α (C/EBPα) that is translated from an alternative non‐AUG initiation codon. We show that a basic amino‐acid motif within its N‐terminus is required for nucleolar retention and for interaction with nucleophosmin (NPM). In the nucleoli, extended‐C/EBPα occupies the ribosomal DNA (rDNA) promoter and associates with the Pol I‐specific factors u pstream‐b inding f actor 1 (UBF‐1) and SL1 to stimulate rRNA synthesis. Furthermore, during differentiation of HL‐60 cells, endogenous expression of extended‐C/EBPα is lost concomitantly with nucleolar C/EBPα immunostaining probably reflecting the reduced requirement for ribosome biogenesis in differentiated cells. Finally, overexpression of extended‐C/EBPα induces an increase in cell size. Altogether, our results suggest that control of rRNA synthesis is a novel function of C/EBPα adding to its role as key regulator of cell growth and proliferation.     

Morphologic evaluation and expression of matrix metalloproteinases-2 and 9 and nitric oxide during experimental periodontal disease in rat

The immunopathologic and inflammatory mechanisms involved in periodontal disease (PD) include the participation of host resident, inflammatory cells and chemical mediators. Metalloproteinases (MMPs) and nitric oxide (NO) play essential role in extracellular matrix turnover of periodontal tissue destruction. In this study, by means of RT-PCR through semi-quantitative densitometric scanning methods, the expression of MMPs -2 and -9 and inducible NO synthase (iNOS) was temporally and spatially investigated during the destructive mechanisms of experimentally induced PD in rats. Samples from different periods were microscopically analyzed and compared with the contralateral side (control). Our results showed significant expression of MMP-9 and iNOS in tissues affected by PD, as compared with controls, three days after PD induction, simultaneously with the beginning of alveolar bone loss. At 7 days post induction, only the MMP-9 mRNA presented a significantly higher expression, as compared with the respective controls. Thus, in the rat ligature-induced PD, MMP-9 and iNOS might importantly participate in the early stages of the disease, including inflammatory cell migration, tissue destruction and alveolar bone resorption. Also, we may suggest that the exuberant presence of PMNs may be related to the important expression of iNOS and MMP-9 found at 3 days post induction.     

HIV-specific antibody among voluntary blood donors in Lower Saxony (FRG)

Anti-HIV test results of the Red Cross Blood Transfusion Service of Lower Saxony from 1 June 1985 to 31 July 1986 inclusive were analysed retrospectively. Nine out of 70,936 donors who had not donated blood before 1 June 1985 (first-time donors) and 9 out of 261,231 donors who had donated blood before this date (repeating donors) were found anti-HIV confirmed positive at the time of the first blood donation during the study period. The prevalence of HIV antibody in first-time donors was significantly higher than in repeating donors (p less than 0.01). It was concluded that some members of risk groups used blood donation to obtain an anti-HIV test result. One out of 30,300 blood donations was confirmed anti-HIV positive. The results of this study justify the transfusion of blood donations that are reactive only in the initial ELISA test.     

When the same is not the same: phenotypic variation reveals different plant ecological strategies within species occurring in distinct Neotropical savanna habitats

Variations in abiotic characteristics such as soil water availability and fertility impose different selective pressures on plant populations. This may produce intraspecific variability in functional traits, even at a fine spatial scale. We investigated whether functional traits related to water-use efficiency, resource-retention strategy, soil nutrient acquisition, and fire tolerance differ in species that occur in two different habitats of Brazilian Cerrado: rocky savannas and savanna woodlands. Rocky savannas occur over sandstone, quartzite outcrops and have shallow nutrient-poor and low-moisture rocky soils, while savanna woodlands occur over well-drained and deep soils with frequent fire regimes. We measured nine functional traits of 40 tree species that occur in both habitats. Rocky savanna individuals exhibited a greater water-use efficiency strategy. The resource-retention strategy in rocky savanna individuals was corroborated by lower adult maximum height. However, despite the lower nutrient availability in rocky savanna soils, we only detected lower leaf phosphorus content in individuals from this habitat. Furthermore, individuals from both habitats had equally thick bark, suggesting that the fire-defense strategy is related to a stable, rather than plastic trait. Overall, our results highlight the central role of contrasting soil water availability patterns in driving phenotypic plasticity within species. We conclude that savanna species are responding to water and nutrient availabilities, via plasticity in traits related to the resource-retention strategy, and preparing for future fires, via uniformly thick bark. Wide plant distribution in contrasting habitats is possible for species that can shift ecological strategies to survive in nutrient- and water-limited habitats such as rocky savannas.     

Essential role of the PSI–LHCII supercomplex in photosystem acclimation to light and/or heat conditions by state transitions

Light and temperature affect state transitions through changes in the plastoquinone (PQ) redox state in photosynthetic organisms. We demonstrated that light and/or heat treatment induced preferential photosystem (PS) I excitation by binding light-harvesting complex II (LHCII) proteins. The photosystem of wheat was in state 1 after dark overnight treatment, wherein PQ was oxidized and most of LHCII was not bound to PSI. At the onset of the light treatment [25 °C in the light (100 µmol photons m^?2 s^?1)], two major LHCIIs, Lhcb1 and Lhcb2 were phosphorylated, and the PSI–LHCII supercomplex formed within 5 min, which coincided with an increase in the PQ oxidation rate. Heat treatment at 40 °C of light-adapted wheat led to further LHCII protein phosphorylation of, resultant cyclic electron flow promotion, which was accompanied by ultrafast excitation of PSI and structural changes of thylakoid membranes, thereby protecting PSII from heat damage. These results suggest that LHCIIs are required for the functionality of wheat plant PSI, as it keeps PQ oxidized by regulating photochemical electron flow, thereby helping acclimation to environmental changes.     

Toxicity of <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> LP1-G Against Susceptible and Resistant <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> and the Cloning of the Mosquitocidal Toxin Gene

Bacillus sphaericus LP1-G, belonging to flagellar serotype H3, has been found to have moderate toxicity against two resistant Culex quinquefasciatus colonies (RLCq1 and RLCq2) and the susceptible contrast (SLCq). With an aim of screening mosquitocidal acting factor, a partial genome library was prepared from a partial HindIII digest of the total DNA from Bacillus sphaericus LP1-G. Two thousand twenty Escherichia coli clones were screened for toxicity against susceptible SLCq, and a toxic clone, designated E-UL68, was chosen for further study. The recombinant E-UL68 performed toxicity against both susceptible and two resistant colonies, having the same level of toxicity as that of wide-type strain LP1-G. Sequence analysis revealed that the inserted fragment was composed of 3876 nucleotides and contained a complete gene, whose sequence was identical to that of the mtx gene from B. sphaericus SSII-1. Because the binary toxin produced during sporulation of strain LP1-G has no activity against the target mosquitoes, this indicates that the Mtx toxin or other active factors might perhaps be responsible for the toxicity of LP1-G against different colonies of mosquito larvae.Received: 7 October 2002 / Accepted: 4 November 2002