GCAP1 rescues rod photoreceptor response in GCAP1/GCAP2 knockout mice

Visual transduction in retinal photoreceptors operates through a dynamic interplay of two second messengers, Ca(2+) and cGMP. Ca(2+) regulates the activity of guanylate cyclase (GC) and the synthesis of cGMP by acting on a GC-activating protein (GCAP). While this action is critical for rapid termination of the light response, the GCAP responsible has not been identified. To test if GCAP1, one of two GCAPs present in mouse rods, supports the generation of normal flash responses, transgenic mice were generated that express only GCAP1 under the control of the endogenous promoter. Paired flash responses revealed a correlation between the degree of recovery of the rod a-wave and expression levels of GCAP1. In single cell recordings, the majority of the rods generated flash responses that were indistinguishable from wild type. These results demonstrate that GCAP1 at near normal levels supports the generation of wild-type flash responses in the absence of GCAP2.     

Regulation of Medicago sativa L. somatic embryogenesis by gibberellins

The influence of exogenous gibberellic acid (GA₃) andpaclobutrazol, an inhibitor of gibberellin biosynthesis, on growth of callusandsomatic embryogenesis in petiole-derived tissue cultures of Medicagosativa L. has been investigated. GA₃ (0.5–500M) or paclobutrazol(5–100 M) were added to either an induction (with 2,4 Dand kinetin) or a differentiation medium (without plant growth regulators).Gibberellin A₃, applied during the induction as well as thedifferentiation stage, reduced the weight of callus and increased the number ofsomatic embryos in Medicago sativa L. tissue cultures.Somatic embryo production was increased more by the presence of exogenousGA₃ in the differentiation than induction medium. The inclusion ofpaclobutrazol in the induction or differentiation medium caused the inhibitionof callus growth and embryo production. Callus growth was much less affectedthan embryogenesis. These results indicate that gibberellins are beneficial forboth embryoinduction and formation. The level of endogenous gibberellins is presumablysufficient for callus induction and growth. However, it seems not optimal forthe induction and particularly for the differentiation of embryos.     

Changes in physico-chemical properties of human large intestine tumour cells membrane

Tumour cells produce and excrete to blood many substances which are present in the cell itself in trace amounts only. Our work has been aimed at the determination of changes in electric charge and in phospholipid composition of large intestine normal mucosa and colorectal cancer cells.Surface charge density of tumour unaffected mucosa and of tissue sections from tumours, was measured by electrophoresis. The measurements were carried out at various pH of solution. Membrane isoelectric point was determined by measuring its electric charge in function of pH as well as total positive charge at low pH and total negative charge at high pH. Qualitative and quantitative composition of phospholipids in the membrane was determined by HPLC. Four phospholipid classes were identified: PI, PS, PE and PC and their surface concentrations were determined.The electric charge calculated from phospholipid concentrations is by three orders of magnitude higher than that determined electrophoretically. It indicates that the groups present in the membrane surface are involved in equilibria in which the charge is neutralized.The electric charge calculated from phospholipid concentrations is by three orders of magnitude higher than that determined electrophoretically. It indicates that the groups present in the membrane surface are involved in equilibria in which the charge is neutralized.Tumour changes provoke an increase in surface charge density of large intestine membrane, whereas the content of individual phospholipids increased or decreased depending on a patient.     

Immunofluorescence stimulated emission depletion microscopy

We report immunofluorescence imaging with a spatial resolution well beyond the diffraction limit. An axial resolution of approximately 50 nm, corresponding to 1/16 of the irradiation wavelength of 793 nm, is achieved by stimulated emission depletion through opposing lenses. We have demonstrated not only that an antibody-tagged label is stable enough to be recorded in this microscopy mode, but also that subdiffraction resolution can be obtained using a standard immunofluorescence preparation.     

Tissue-specific expression of Pa18, a putative lipid transfer protein gene,during embryo development in Norway spruce (Picea abies)

A full-length Picea abies cDNA clone Pa18, encoding a protein with the characteristics of plant lipid transfer proteins, has been isolated and characterized. The size of the deduced 173 amino acid (aa) long protein is around 18 kDa. The first 100–120 aa show similarity to angiosperm lipid transfer proteins in amino acid sequence as well as in predicted secondary structure. The Pa18 gene is constitutively expressed in embryogenic cultures of Picea abies representing different stages of development as well as in non-embryogenic callus and seedlings. The Pa18 gene product has an antimicrobial activity. In situ hybridization showed that the Pa18 gene is equally expressed in all embryonic cells of proliferating embryogenic cultures but during embryo maturation the expression of the gene in maturing and mature somatic as well as in mature zygotic embryos is stronger in the outer cell layer than in other tissues. Southern blot analysis at different stringencies was consistent with a single gene with one or two copies rather than a gene family. Twenty independent transgenic sublines over- and under-expressing the Pa18 gene under the Zea mays ubiquitin promoter were established. There was a high yield of mature somatic embryos with a smooth surface only in untransformed, control cultures. Irrespective of the expression level of Pa18, the somatic embryos started to mature when given a maturation treatment. However, in the transgenic sublines, the outer cells in the maturing embryos frequently became elongated and vacuolated instead of remaining small and uniform. One explanation for this was that the expression of Pa18 was not restricted to the outer cell layer in transformed sublines. Angiosperms and gymnosperms separated about 300 million years ago and the embryo genesis is different in the two groups. The outer cell layer (protoderm), the first tissue to differentiate, is less clearly delineated in gymnosperms. For normal embryo development in angiosperms, expression of the LTP gene must be restricted to the protodermal cells. In this work we show that the expression of the Pa18 gene must be restricted to the putative protodermal cells of the gymnosperm.     

High Viral Loads of Epstein-Barr Virus DNA in Peripheral Blood of Patients with Chronic Lymphocytic Leukemia Associated with Unfavorable Prognosis

Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus that infects more than 90% of the world population. The potential involvement of EBV in the clinical course of chronic lymphocytic leukemia (CLL) remains unexplained. The aim of this study was to determine whether EBV-DNA load in the peripheral blood mononuclear cells (PBMCs) of CLL patients may influence heterogeneity in the course of the disease. The study included peripheral blood samples from 115 previously untreated patients with CLL (54 women and 61 men) and 40 healthy controls (16 women and 24 men). We analyzed the association between the EBV-DNA load in PBMCs and the stage of the disease, adverse prognostic factors, and clinical outcome. Detectable numbers of EBV-DNA copies in PBMCs were found in 62 out of 115 CLL patients (53.91%). The EBV-DNA copy number/μg DNA was significantly higher in patients who required early implementation of treatment, presented with lymphocyte count doubling time <12 months, displayed CD38-positive or ZAP-70-positive phenotype, and with the del(11q22.3) cytogenetic abnormality. Furthermore, the EBV-DNA copy number/μg DNA showed significant positive correlation with the concentrations of lactate dehydrogenase (LDH) and beta-2-microglobulin. We have shown that in CLL patients, higher EBV-DNA copy number predicted shorter survival and shorter time to disease progression, and it was associated with other established unfavorable prognostic factors. This suggests that EBV may negatively affect the outcome of CLL.     

Structure and Ultrastructure of the Endodermal Region of the Alimentary Tract in the Freshwater Shrimp Neocaridina heteropoda (Crustacea,Malacostraca)

The freshwater shrimp Neocaridina heteropoda (Crustacea, Malacostraca, Decapoda) originates from Asia and is one of the species that is widely available all over the world because it is the most popular shrimp that is bred in aquaria. The structure and the ultrastructure of the midgut have been described using X-ray microtomography, transmission electron microscopy, light and fluorescence microscopes. The endodermal region of the alimentary system in N. heteropoda consists of an intestine and a hepatopancreas. No differences were observed in the structure and ultrastructure of males and females of the shrimp that were examined. The intestine is a tube-shaped organ and the hepatopancreas is composed of two large diverticles that are divided into the blind-end tubules. Hepatopancreatic tubules have three distinct zones – proximal, medial and distal. Among the epithelial cells of the intestine, two types of cells were distinguished – D and E-cells, while three types of cells were observed in the epithelium of the hepatopancreas – F, B and E-cells. Our studies showed that the regionalization in the activity of cells occurs along the length of the hepatopancreatic tubules. The role and ultrastructure of all types of epithelial cells are discussed, with the special emphasis on the function of the E-cells, which are the midgut regenerative cells. Additionally, we present the first report on the existence of an intercellular junction that is connected with the E-cells of Crustacea.     

Antibodies against Immature Virions Are Not a Discriminating Factor for Dengue Disease Severity

Humoral immunity plays an important role in controlling dengue virus (DENV) infection. Antibodies (Abs) developed during primary infection protect against subsequent infection with the same dengue serotype, but can enhance disease following secondary infection with a heterologous serotype. A DENV virion has two surface proteins, envelope protein E and (pre)-membrane protein (pr)M, and inefficient cleavage of the prM protein during maturation of progeny virions leads to the secretion of immature and partially immature particles. Interestingly, we and others found that historically regarded non-infectious prM-containing DENV particles can become highly infectious in the presence of E- and prM-Abs. Accordingly, we hypothesized that these virions contribute to the exacerbation of disease during secondary infection. Here, we tested this hypothesis and investigated the ability of acute sera of 30 DENV2-infected patients with different grades of disease severity, to bind, neutralize and/or enhance immature DENV2. We found that a significant fraction of serum Abs bind to the prM protein and to immature virions, but we observed no significant difference between the disease severity groups. Furthermore, functional analysis of the Abs did not underscore any specific correlation between the neutralizing/enhancing activity towards immature DENV2 and the development of more severe disease. Based on our analysis of acute sera, we conclude that Abs binding to immature virions are not a discriminating factor in dengue pathogenesis.     

New Insights into Plagiogrammaceae (Bacillariophyta) Based on Multigene Phylogenies and Morphological Characteristics with the Description of a New Genus and Three New Species

Plagiogrammaceae, a poorly described family of diatoms, are common inhabitants of the shallow marine littoral zone, occurring either in the sediments or as epiphytes. Previous molecular phylogenies of the Plagiogrammaceae were inferred but included only up to six genera: Plagiogramma, Dimeregramma, Neofragilaria, Talaroneis, Psammogramma and Psammoneis. In this paper, we describe a new plagiogrammoid genus, Orizaformis, obtained from Bohai Sea (China) and present molecular phylogenies of the family based on three and four genes (nuclear-encoded large and small subunit ribosomal RNAs and chloroplast-encoded rbcL and psbC). Also included in the new phylogenies is Glyphodesmis. The phylogenies suggest that the Plagiogrammaceae is composed of two major clades: one consisting of Talaroneis, Orizaformis and Psammoneis, and the second of Glyphodesmis, Psammogramma, Neofragilaria, Dimeregramma and Plagiogramma. In addition, we describe three new species within established genera: Psammoneis obaidii, which was collected from the Red Sea, Saudi Arabia; and Neofragilaria stilus and Talaroneis biacutifrons from the Mozambique Channel, Indian Ocean, and illustrate two new combination taxa: Neofragilaria anomala and Neofragilaria lineata. Our observations suggest that the biodiversity of the family is strongly needed to be researched, and the phylogenetic analyses provide a useful framework for future studies of Plagiogrammaceae.