The use of an internal standard for semiquantitative analysis of low temperature (77°K) fluorescence of photosynthetic cells

A simple method is described which permits quantitative estimation of chlorophyll fluorescence emission intensity relative to the chlorophyll concentration in sample material at 77°K. A fluorescent standard excited by light of a similar wavelength as that of the sample material but emitting at a slightly shorter wavelength is mixed with the sample in known proportions. The fluorescence yield of the various peaks of the sample are normalized to the fluorescence yield of the internal standard. The spectra are recorded using an inexpensive attachment consisting of a Dewar holder, mounted instead of the light source of any double beam or split beam spectrophotometer. A glass rod is immersed in the sample standard mixture and then placed in the liquid nitrogen containing Dewar. Exciting light is provided by a light pipe mounted at 90° to the sample holder, connected with a tungsten-hallogen lamp provided with an appropriate filter. This method has been found to be particularly useful for the quantitation of chlorophyll fluorescence emission at 77°K of photosynthetic cells or chlorophyll containing membrane fractions. For this purpose, phycocyanin was found to be a suitable internal standard.     

Changes in thylakoid polypeptide phosphorylation during membrane biogenesis in Chlamydomonas reinhardii y-1

Thylakoid polypeptide phosphorylation has been studied in vivo and in vitro during plastid differentiation in Chlamydomonas reinhardii y-1. Pulse labeling cells at different stages of greening with [³²P]orthophosphate revealed differences in the pattern of protein phosphorylation. In the early phase of greening the 44–47 kDa reaction center II polypeptides were labeled but the 22–24 kDa polypeptides of the light-harvesting chlorophyll $\text{a}\text{b-}\text{protein}$ complex (LHC) were not. Later in the greening, coinciding with the formation of the antenna of Photosystem I and membrane stacking, the converse was found. Furthermore, the 22–24 kDa polypeptides of grana lamellae were less labeled than the same polypeptides found in the corresponding stroma lamellae. Polypeptides in the molecular mass range of 32–34 kDa were phosphorylated at all stages following the onset of greening. Dark-grown cells did not incorporate ³²P in vivo or in vitro into the polypeptides present in the residual thylakoids. Similarly, cells greened in the presence of chloramphenicol, in which the synthesis of reaction centers is inhibited, showed no light-stimulated phosphorylation in vitro. However, the residual 32–34 kDa and 44–47 kDa polypeptides found in thylakoids of these cells were phosphorylated in vivo, whereas the LHC polypeptides synthesized in the presence of chloramphenicol were not. Phosphorylation of the LHC polypeptides (22–24 kDa) in these cells occurred if new reaction center polypeptides and all antennae components were formed, following removal of the inhibitor and further incubation of the cells in the light. Phosphorylation of LHC polypeptides was not resumed if active reaction centers were formed in the absence of complete restoration of all antenna components (incubation in the dark or light with addition of cycloheximide). It is concluded that phosphorylation is correlated with the thylakoid polypeptide content and organization.     

The Prawn Macrobrachium vollenhovenii in the Senegal River Basin: Towards Sustainable Restocking of All-Male Populations for Biological Control of Schistosomiasis

Early malacological literature suggests that the outbreak of schistosomiasis, a parasitic disease transmitted by aquatic snails, in the Senegal River basin occurred due to ecological changes resulting from the construction of the Diama dam. The common treatment, the drug praziquantel, does not protect from the high risk of re-infection due to human contact with infested water on a daily basis. The construction of the dam interfered with the life cycle of the prawn Macrobrachium vollenhovenii by blocking its access to breeding grounds in the estuary. These prawns were demonstrated to be potential biological control agents, being effective predators of Schistosoma-susceptible snails. Here, we propose a responsible restocking strategy using all-male prawn populations which could provide sustainable disease control. Male prawns reach a larger size and have a lower tendency to migrate than females. We, therefore, expect that periodic restocking of all-male juveniles will decrease the prevalence of schistosomiasis and increase villagers'' welfare. In this interdisciplinary study, we examined current prawn abundance along the river basin, complemented with a retrospective questionnaire completed by local fishermen. We revealed the current absence of prawns upriver and thus demonstrated the need for restocking. Since male prawns are suggested to be preferable for bio-control, we laid the molecular foundation for production of all-male M. vollenhovenii through a complete sequencing of the insulin-like androgenic gland-encoding gene (IAG), which is responsible for sexual differentiation in crustaceans. We also conducted bioinformatics and immunohistochemistry analyses to demonstrate the similarity of this sequence to the IAG of another Macrobrachium species in which neo-females are produced and their progeny are 100% males. At least 100 million people at risk of schistosomiasis are residents of areas that experienced water management manipulations. Our suggested non-breeding sustainable model of control—if proven successful—could prevent re-infections and thus prove useful throughout the world.     

Improved Bacterial Detection Using Immobilized Acyl-Lysyl Oligomers

The global need to improve bacterial detection in liquid media has motivated multidisciplinary research efforts toward developing new approaches that overcome the shortcomings of traditional techniques. We recently proposed the use of oligomers of acylated lysyls (OAKs) in their resin-linked form (ROAKs) for the efficient, robust, and inexpensive filtration of bacteria. Here, to investigate the potential for the use of ROAKs in downstream applications, we first examined the capacity of ROAKs to capture bacteria as a function of environmental conditions and structure-activity relationships (SARs). We next assessed their ability to release the captured bacteria and then combined both abilities to improve real-time PCR outcomes. ROAKs were able to deplete liquid samples of bacterial content after incubation or continuous flow, illustrating the efficient capture of different bacterial species under a wide range of ionic strength and pH conditions. We also show circumstances for the significant release of captured bacteria, live or dead, for further analysis. Finally, the SAR study revealed a shorter ROAK derivative exhibiting a capture capacity similar to that of the parent construct but the increased recovery of ROAK-bound bacteria, enabling improvement of the detection sensitivity by 20-fold. Collectively, the data support the potential usefulness of a simple, robust, and efficient approach for rapid capture/analysis of bacteria from tap water and, possibly, from more complex media.