Fluorescence correlation spectroscopy as a sensitive and useful tool for revealing potential overlaps between the epitopes of monoclonal antibodies on viral particles

Although the enzyme-linked immunosorbent assay (ELISA) is well established for quantitating epitopes on inactivated virions used as vaccines, it is less suited for detecting potential overlaps between the epitopes recognized by different antibodies raised against the virions. We used fluorescent correlation spectroscopy (FCS) to detect the potential overlaps between 3 monoclonal antibodies (mAbs 4B7-1H8-2E10, 1E3-3G4, 4H8-3A12-2D3) selected for their ability to specifically recognize poliovirus type 3. Competition of the Alexa488-labeled mAbs with non-labeled mAbs revealed that mAbs 4B7-1H8-2E10 and 4H8-3A12-2D3 compete strongly for their binding sites on the virions, suggesting an important overlap of their epitopes. This was confirmed by the cryo-electron microscopy (cryo EM) structure of the poliovirus type 3 complexed with the corresponding antigen-binding fragments (Fabs) of the mAbs, which revealed that Fabs 4B7-1H8-2E10 and 4H8-3A12-2D3 epitopes share common amino acids. In contrast, a less efficient competition between mAb 1E3-3G4 and mAb 4H8-3A12-2D3 was observed by FCS, and there was no competition between mAbs 1E3-3G4 and 4B7-1H8-2E10. The Fab 1E3-3G4 epitope was found by cryoEM to be close to but distinct from the epitopes of both Fabs 4H8-3A12-2D3 and 4B7-1H8-2E10. Therefore, the FCS data additionally suggest that mAbs 4H8-3A12-2D3 and 4B7-1H8-2E10 bind in a different orientation to their epitopes, so that only the former sterically clashes with the mAb 1E3-3G4 bound to its epitope. Our results demonstrate that FCS can be a highly sensitive and useful tool for assessing the potential overlap of mAbs on viral particles.     

Hereditary persistence of fetal hemoglobin, beta thalassemia, and the hemoglobin delta-beta locus: further family data and genetic interpretations.

Three Negro kindreds with hereditary persistence of fetal hemoglobin (HPFH) alone and in combination with various other hemoglobin abnormalities including beta thalassemia are presented. Among 11 offspring of two women heterozygous for both HPFH and the delta chain mutation Hb B2, five inherited the HPFH gene and six inherited the Hb B2 gene. In another kindred, a man inferred to be heterozygous for both HPFH and Hb C had six children; three offsprivg obtained the Hb C gene and three the HPFH gene. Similarly, a woman heterozygous for both Hb S and HPFH transmitted the Hb S gene to one of her two children and the HPFH gene to the other. Thus among 19 offspring, no crossovers between the HPFH locus or the Hb delta-beta locus were observed. These and earlier data are compatible with deletion of the Hb beta and delta loci as the primary event to explain the genetic origin of HPFH. Genetic considerations indicate that the finding of a single person with a hematologically normal phenotype among offspring of heterozygotes for both the African type of HPFH and a Hb beta or Hb delta structural abnormality would invalidate the deletion model.     

Is mucus involved in biocrystallization?

Summary Freeze-dried intestinal mucus of sea-water-adapted eels was analysed by scanning electron microscopy (SEM) and X-ray microanalysis. Calcite crystals were observed in the mucus fibres; their concentration increased along the hindgut. Random SEM observations made in situ indicated that mucus fibres were involved in the genesis of these crystals. Calcium-rich mucus globules were found fused inside crystal matrices. Single typical rhombohedric crystals of various complexity appeared within the mucus framework. The steps of crystal biogenesis were reconstituted in in-vitro conditions.     

Cell cycle regulation of histone H1O in CHO cells: a flow cytofluorimetric study after double staining of the cells

The regulation of histone H1O content throughout the cell cycle of non-synchronized Chinese hamster ovary (CHO) cells has been studied using double fluorescent staining and flow cytofluorometry. In exponentially growing cells, the amount of H1O was found to be proportional to the DNA content of the cells, indicating that the protein is synthesized throughout the cell cycle. However, when cells were arrested in G1 at saturation density the amount of H1O was greater than that found in G1 cells of the exponentially growing population. In contrast, the levels of H1-1 were the same for G1 cells of both populations. These results show that the regulation of H1O accumulation differs from that of other histones.     

Calcium-induced phosphorylations and [125I]calmodulin binding in renal membrane preparations

Calcium-induced phosphorylated intermediates and calmodulin-binding proteins in membrane preparations from th renal cortex were analyzed by SDS-polyacrylamide gel electrophoresis at low pH, protein electroblotting and [¹²⁵I]calmodulin overlay. Two calcium-induced phosphoproteins were found, with a molecular mass of 135 and 115 kDa, respectively. By comparing different preparations characterized by marker enzymes, it was shown that the 135 kDa phosphoprotein is localized in the basal-lateral fragment of the plasma membrane, whereas the 115 kDa phosphoprotein is more pronounced in preparations containing a high proportion of endoplasmic reticulum. A prominent calmodulin-binding protein comigrated with the 135 kDa phosphoprotein; there was no calmodulin binding to polypeptides in the molecular mass range of the 115 kDa phosphoprotein. Partial proteolysis by trypsin and the effect of $\text{20 μ}\text{M La}{}^{\mathrm{2+}}$ on the formation of phosphoproteins before and after trypsinization support the conclusion that the 135 kDa protein can be identified with the plasma membrane calcium pump, whereas the 115 kDa phosphoprotein is the phosphorylated intermediate of a different type of calcium pump probably originating from the endoplasmic reticulum. Calmodulin binding in renal membrane preparations analyzed on Laemmli-type slab gels revealed that there are many calmodulin-binding proteins in our preparations. We have identified one band with the renal calcium pump localized in the basal-lateral membrane. Another calmodulin-binding protein migrating at 108 kDa, is not localized in the basal-lateral membrane and could be one of the calmodulin-binding proteins originating from the cytoskeleton.     

Life cycle assessment of two baby food packaging alternatives: glass jars vs. plastic pots

Background, aim, and scope  This paper compares the life cycle assessment (LCA) of two packaging alternatives used for baby food produced by Nestlé: plastic pot and glass jar. The study considers the environmental impacts associated with packaging systems used to provide one baby food meal in France, Spain, and Germany in 2007. In addition, alternate logistical scenarios are considered which are independent of the two packaging options. The 200-g packaging size is selected as the basis for this study. Two other packaging sizes are assessed in the sensitivity analysis. Because results are intended to be disclosed to the public, this study underwent a critical review by an external panel of LCA experts. Materials and methods  The LCA is performed in accordance to the international standards ISO 14040 and ISO 14044. The packaging systems include the packaging production, the product assembly, the preservation process, the distribution, and the packaging end-of-life. The production of the content (before preservation process), as well as the use phase are not taken into account as they are considered not to change when changing packaging. The inventory is based on data obtained from the baby food producer and the suppliers, data from the scientific literature, and data from the ecoinvent database. Special care is taken to implement a system expansion approach for end-of-life open and closed loop recycling and energy production (ISO 14044). A comprehensive impact assessment is performed using two life cycle impact assessment methodologies: IMPACT 2002+ and CML 2001. An extensive uncertainty analysis using Monte Carlo as well as an extensive sensitivity study are performed on the inventory and the reference flows, respectively. Results  When looking at the impacts due to preservation process and packaging (considering identical distribution distances), we observe a small but significant environmental benefit of the plastic pot system over the glass jar system. Depending on the country, the impact is reduced by 14% to 27% for primary energy, 28% to 31% for global warming, 31% to 34% for respiratory inorganics, and 28% to 31% for terrestrial acidification/nutrification. The environmental benefit associated with the change in packaging mainly results from (a) production of plastic pot (including its end-of-life; 43% to 51% of total benefit), (b) lighter weight of packaging positively impacting transportation (20% to 35% of total benefit), and (c) new preservation process permitted by the plastic system (23% to 34% of total benefit). The jar or pot (including cap or lid, cluster, stretch film, and label) represents approximately half of the life cycle impacts, the logistics approximately one fourth, and the rest (especially on-site energy, tray, and hood) one fourth. Discussion  The sensitivity analysis shows that assumptions made in the basic scenarios are rather conservative for plastic pots and that the conclusions for the 200-g packaging size also apply to other packaging sizes. The uncertainty analysis performed on the inventory for the German market situation shows that the plastic pot system has less impact than the glass jar system while considering similar distribution distances with a confidence level above 97% for most impact categories. There is opportunity for further improvement independent of the type of packaging used, such as by reducing distribution distances while still optimizing lot size. The validity of the main conclusions presented in this study is confirmed by results of both impact assessment methodologies IMPACT 2002+ and CML 2001. Conclusions  For identical transportation distances, the plastic pot system shows a small but significant reduction in environmental burden compared to the glass jar system. Recommendations and perspectives  As food distribution plays an important role in the overall life cycle burdens and may vary between scenarios, it is important to avoid additional transportation of the packaged food in order to maintain or even improve the advantage of the plastic pot system. The present study focuses on the comparison of packaging systems and directly related consequences. It is recommended that further environmental optimization of the product also includes food manufacturing (before preservation process) and the supply chain of raw materials.     

Uncoupled IP3 receptor can function as a Ca2+-leak channel: cell biological and pathological consequences

Ca(2+) release via intracellular release channels, IP(3)Rs (inositol 1,4,5-trisphosphate receptors) and RyRs (ryanodine receptors), is perhaps the most ubiquitous and versatile cellular signalling mechanism, and is involved in a vast number of cellular processes. In addition to this classical release pathway there is limited, but yet persistent, information about less well-defined Ca(2+)-leak pathways that may play an important role in the control of the Ca(2+) load of the endo(sarco)plasmic reticulum. The mechanisms responsible for this 'basal' leak are not known, but recent data suggest that both IP(3)Rs and RyRs may also operate as Ca(2+)-leak channels, particularly in pathological conditions. Proteolytic cleavage or biochemical modification (such as hyperphosphorylation or nitrosylation), for example, occurring during conditions of cell stress or apoptosis, can functionally uncouple the cytoplasmic control domains from the channel domain of the receptor. Highly significant information has been obtained from studies of malfunctioning channels in various disorders; for example, RyRs in cardiac malfunction or genetic muscle diseases and IP(3)Rs in neurodegenerative diseases. In this review we aim to summarize the existing information about functionally uncoupled IP(3)R and RyR channels, and to discuss the concept that those channels can participate in Ca(2+)-leak pathways.