Formin-like 1 (FMNL1) Is Regulated by N-terminal Myristoylation and Induces Polarized Membrane Blebbing

The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1γ) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD). FMNL1γ is specifically located at the cell membrane and cortex in diverse cell lines. Similar localization of FMNL1 was observed for a mutant lacking the DAD domain (FMNL1ΔDAD), indicating that deregulation of autoinhibition is effective in FMNL1γ. Expression of both FMNL1γ and FMNL1ΔDAD induces polarized nonapoptotic blebbing that is dependent on N-terminal myristoylation of FMNL1 but independent of Src and ROCK activity. Thus, our results describe N-myristoylation as a regulative mechanism of FMNL1 responsible for membrane trafficking potentially involved in a diversity of polarized processes of hematopoietic lineage-derived cells.     

Dark starvation and chloroplast function

Summary Mature spinach plants were held in the dark for several days. The photochemical activities and the activity of some enzymes related to NADP reduction were follwed in the chloroplasts isolated from leaves after dark starvation. Photosystem-II, measured by reduction of DPIP, remained stable during 6 days of darkening. The decrease of NADP reduction which appeared after 2 days of starvation was found to be due to protein autolysis rather than inactivation of the photosystems. The stability of photosystem-I was demonstrated by reactivation of NADP reduction after addition of purified ferredoxin and ferredoxin-NADP-reductase. After 4 days of starvation the restoration of the NADP reduction required in addition another, low-molecular-weight factor. From the isolation procedure and from its properties this factor is assumed to be identical with FRS. However, even in the presence of FRS only half of the total activity is restored after 7 days. The activity of the NADP-reducing system is restored in vivo when plants kept for 7 days in the dark are again illuminated.Abbreviations NADP nicotinamide-adenine-dinucleotide phosphate - DPIP 2,6-dichlorophenolindophenol - DCMU (3,4-dichlorophenyl)-1,1-dimethylurea - FRS ferredoxin-reducing-substance     

Expression of human asparagine synthetase in Escherichia coli

Human asparagine synthetase was expressed in Escherichia coli. Synthesis of the enzyme was demonstrated by immunoblotting and by complementation of asparagine auxotrophy in E. coli. The recombinant enzyme was shown to have both the ammonia- and glutamine-dependent asparagine synthetase activity in vitro. Compared to asparagine synthetase isolated from beef pancreas, the one expressed in E. coli migrated at a slightly slower rate on a denaturing protein gel. In contrast with previous reports, the data obtained here strongly suggest that the active enzyme is a homodimer. The production of soluble and active enzyme was shown to be highly temperature-dependent. Expression at 37 degrees C yielded no soluble enzyme, whereas growth at 30 and 21 degrees C favored the production of soluble asparagine synthetase. The incubation temperature was also important for complementation of asparagine auxotrophy in E. coli, as growth in the absence of asparagine occurred at 30 degrees C and not at 37 degrees C.     

Development of the visual system of anchovy larvae,Engraulis anchoita: A microanatomical description

During the early ontogeny of fish larvae, the accurate development of the visual system plays a key role, because it is involved in locating food, orientation, selection of favorable habitat, and evasion of predators. The structure of the eye of the fish is typical of vertebrates, with some modifications related to the aquatic environment. In the present work, we describe the development of the larval eye of Engraulis anchoita for the first time. Larvae were collected at the Permanent Station of Environmental Studies (EPEA) in coastal waters of the Southwestern Atlantic Ocean during research cruises in 2015 and 2016. We describe the histology of the retina layers, determine the beginning of the functionality of the eye, and discuss a possible synchronization with the development of the digestive tract. This study provides information about the biology of E. anchoita, the most abundant fish species in the southwestern Atlantic Ocean. Also, recent studies have shown responses of the retina and other tissues to the increase in environmental acidity. Therefore, results of this study are also discussed with respect to the possible effect of acidification on the larvae of this species. The continuity of the time series developed at the EPEA will allow monitoring the effect of long-term environmental and biological variables on the early ontogeny of anchovy in the context of climate change. The high commercial fishing potential of E. anchoita due to its high abundance, as well as its essential role in the trophic web of other commercially valuable fishing resources of Argentina, reinforce the need to continue deepening knowledge about this species. Research highlights:

• Eyes of Engraulis anchoita larvae are functional from early larval stages.
• At hatching, the retina is formed by only few layers from which the other layers differentiates during ontogeny.
• Focal distance increases with larval growth.

     

Overproduction, preparation of monoclonal antibodies and purification of E. coli asparagine synthetase A.

In order to explore the structure--function relationship of the Escherichia coli asparagine synthetase A it was necessary to devise a system for overexpression of the gene and purification of the gene product. The E. coli asparagine synthetase A structural gene was fused to the 3' end of the human carbonic anhydrase II structural gene and overexpressed in E. coli. The gene product, a 66 kDa fusion protein, which exhibited asparagine synthetase activity, was purified in a single step by affinity chromatography and used as the antigen for the production of monoclonal antibodies. The monoclonal antibodies were screened by ELISA. Colonies were chosen which were positive for purified fusion protein and negative for purified human carbonic anhydrase II. The E. coli asparagine synthetase A gene was then overexpressed and the gene product was used without purification for the final screen. The antibodies selected were used for immunoaffinity chromatography to purify the recombinant overexpressed E. coli asparagine synthetase A. Thus, a procedure is now available so that asparagine synthetase A can be purified to homogeneity in a single step.     

Obtaining a High Percentage of Explants with Negative Serological Reactions against viruses by Combining Potato Meristem Culture with Antiphytoviral Chemotherapy

A high percentage of plantlets, in which PVX, PVM, and PVS were no longer detectable by precipitin tests was obtained from 100% virus-infected stocks of the potato varieties Rosa and Priobskij rannija by combining meristem (tip) culture with antiphytoviral chemotherapy. When 2,4 - dioxohexahydro 1,3,5-triazinc (DHT) was added to the nutrient media, no virus was detectable serologically in 72% of the corresponding explants of the variety Rosa and in 66% of that of the variety Priobskij rannija, respectively. Contrary to this, all untreated control explants proved virusinfected. Treatment with 2-thiouracil resulted in 46.6%, with cyanoguanidine in 28.6% of plantlets, in which no virus was detectable serologically.     

SuperSAGE analysis of the <Emphasis Type="Italic">Nicotiana attenuata</Emphasis> transcriptome after fatty acid-amino acid elicitation (FAC): identification of early mediators of insect responses

Background  

Plants trigger and tailor defense responses after perception of the oral secretions (OS) of attacking specialist lepidopteran larvae. Fatty acid-amino acid conjugates (FACs) in the OS of the Manduca sexta larvae are necessary and sufficient to elicit the herbivory-specific responses in Nicotiana attenuata, an annual wild tobacco species. How FACs are perceived and activate signal transduction mechanisms is unknown.