One-carbon metabolism in plants. Regulation of tetrahydrofolate synthesis during germination and seedling development

Tetrahydrofolate (THF) is a central cofactor for one-carbon transfer reactions in all living organisms. In this study, we analyzed the expression of dihydropterin pyrophosphokinase-dihydropteroate synthase (HPPK-DHPS) in pea (Pisum sativum) organs during development, and so the capacity to synthesize dihydropteroate, an intermediate in the de novo THF biosynthetic pathway. During seedling development, all of the examined organs/tissues contain THF coenzymes, collectively termed folate, and express the HPPK-DHPS enzyme. This suggests that each organ/tissue is autonomous for the synthesis of THF. During germination, folate accumulates in cotyledons and embryos, but high amounts of HPPK-DHPS are only observed in embryos. During organ differentiation, folate is synthesized preferentially in highly dividing tissues and in photosynthetic leaves. This is associated with high levels of the HPPK-DHPS mRNA and protein, and a pool of folate 3- to 5-fold higher than in the rest of the plant. In germinating embryos and in meristematic tissues, the high capacity to synthesize and accumulate folate correlates with the general resumption of cell metabolism and the high requirement for nucleotide synthesis, major cellular processes involving folate coenzymes. The particular status of folate synthesis in leaves is related to light. Thus, when illuminated, etiolated leaves gradually accumulate the HPPK-DHPS enzyme and folate. This suggests that folate synthesis plays an important role in the transition from heterotrophic to photoautotrophic growth. Analysis of the intracellular distribution of folate in green and etiolated leaves indicates that the coenzymes accumulate mainly in the cytosol, where they can supply the high demand for methyl groups.     

Characterization of sterol uptake in leaf tissues of sugar beet

The uptake of cholesterol has been characterized in leaf discs from mature leaves of sugar beet (Beta vulgaris L.). This transport system exhibited a simple saturable phase with an apparent Michaelis constant ranging from 30 to 190 M depending on the sample. When present at 10 M excess, other sterols were able to inhibit cholesterol uptake. Moreover, binding assays demonstrated the presence of high-affinity binding sites for cholesterol in purified plasma membrane vesicles. In the range 1–60 M, cholesterol uptake showed an active component evidenced by action of the protonophore carbonyl cyanide m-chlorophenylhydrazone. Energy was required as shown by the inhibition of uptake induced by respiration inhibitors (NaN₃), darkness and photosynthesis inhibitors [3-(3,4-dichlorophenyl)-1,1-dimethylurea, methyl viologen]. Moreover, the process was strongly dependent on the experimental temperature. Uptake was optimal at acidic pH (4.0), sensitive to ATPase modulators, inhibited by thiol reagents (N-ethylmaleimide, p-chloromercuribenzenesulfonic acid, Mersalyl) and by the histidyl-group reagent diethyl pyrocarbonate. The addition of cholesterol did not modify H⁺ flux from tissues, indicating that H⁺-co-transport was unlikely to be involved. MgATP did not increase the uptake, arguing against involvement of an ABC cassette-type transporter. By contrast, cryptogein, a sterol carrier protein from the Oomycete Phytophtora cryptogea, greatly increased absorption. Taken together, the results reported in this work suggest that plant cells contain a specific plasma membrane transport system for sterols.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - PCMBS p-chloromercuribenzenesulfonic acid - PMV plasma membrane vesicle - TLC thin-layer chromatography     

The optimal sequence of microvascular repair during prolonged clamping in free flap transfer

During free flap transfer, the surgeon may decide to begin with repair of the artery or the vein(s) and to unclamp the first vessel as soon as repair is completed or maintain the clamping of both vessels until completion of all repairs. Complications can lead to prolonged clamping times, potentially increasing the risk of tissue ischemia, vascular damage, and thrombosis. The goals of the present study were to determine whether the sequence of vessel repair and the duration of clamping affect the success of free flap transfer in cases requiring prolonged clamping. Sixty abdominal fasciocutaneous free flaps based on the superficial inferior epigastric vessels were created in Sprague-Dawley rats. To model clinical situations in which prolonged clamping is necessary, the study used a 1-hour delay before the repair of the second vessel. Flaps were randomized into four groups. In group I (n = 15), the artery was repaired first, and the arterial clamp was removed immediately to allow arterial inflow. In group II (n = 15), the arterial repair was first, and the arterial clamp was maintained until completion of venous repair. In group III (n = 15), venous repair was first, with venous clamping maintained until completion of the arterial repair. In group IV (n = 15), initial venous repair was followed by immediate unclamping, before arterial repair. On release of all clamps, the patency of arteries and veins was confirmed immediately and after 1 hour using a "milking" test. On the fifth postoperative day, each flap was assessed for necrosis and for patency of the anastomoses. Of 15 flaps in each group, five (33 percent) failed in group I, four (27 percent) failed in groups II and III, and six (40 percent) failed in group IV. Differences between groups were not statistically significant (p = 0.8). These results demonstrate that in cases requiring prolonged occlusive clamping (2 to 3 hours), factors such as venous congestion, possible clamp injury, and presence of static blood in contact with the new anastomosis have relatively equivalent contributions to the risk of failure. Accordingly, no advantage seems to be gained by beginning with the artery or the vein or by using early or delayed unclamping of the first vessel repaired.     

Adjacent and spontaneous neurotization after distal digital replantation in children

In an exclusively pediatric population, this retrospective study examined the functional and aesthetic results after distal replantation without nerve suture. The aim was to demonstrate, in the child, the presence of spontaneous nervous regeneration resulting in a fingertip pulp with discriminatory sensation. Eight amputations in eight children with a mean age of 9 years and 2 months on the day of the accident were reviewed. The cases were managed by a single surgeon over a period of 8 years and were collected from two different hand centers. The patients were then examined by a different surgeon, and the data were collected. Sensibility was evaluated using the Weber, Semmes-Weinstein, and wrinkle tests. The results were excellent, with mean values of 4.6 mm for the Weber test, 3.3 for the Semmes-Weinstein test, and a positive wrinkle test in all subjects. All patients thus recovered discriminatory sensation with minimal aesthetic sequelae. The usual factors adversely affecting the results of the replantation (ischemic time, level and mechanism of the amputation, and quality of the venous return) were examined, but no statistical analysis was performed because of the small sample size. This study demonstrates the presence of the clinical phenomenon of adjacent neurotization in the absence of nerve repair. It thus confirms that children are excellent candidates for replantation of the distal extremities, even when nerve suture is not performed.     

Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3)

Secretion of cytolytic granules content at the immunological synapse is a highly regulated process essential for lymphocyte cytotoxicity. This process requires the rapid transfer of perforin containing lytic granules to the target cell interface, followed by their docking and fusion with the plasma membrane. Defective cytotoxicity characterizes a genetically heterogeneous condition named familial hemophagocytic lymphohistiocytosis (FHL), which can be associated with perforin deficiency. The locus of a perforin (+) FHL subtype (FHL3), observed in 10 patients, was mapped to 17q25. This region contains hMunc13-4, a member of the Munc13 family of proteins involved in vesicle priming function. HMunc13-4 mutations were shown to cause FHL3. HMunc13-4 deficiency results in defective cytolytic granule exocytosis, despite polarization of the secretory granules and docking with the plasma membrane. Expressed tagged hMunc13-4 localizes with cytotoxic granules at the immunological synapse. HMunc13-4 is therefore essential for the priming step of cytolytic granules secretion preceding vesicle membrane fusion.     

Consensus-degenerate hybrid oligonucleotide primers for amplification of priming glycosyltransferase genes of the exopolysaccharide locus in strains of the Lactobacillus casei group

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3' degenerate core based on four highly conserved amino acids and a longer 5' consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS(-)) and EPS-producing (EPS(+)) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS(+) bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS(+) strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.     

Fluorescence-activated cell sorting of specific affibody-displaying staphylococci

Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.     

Metagenome survey of biofilms in drinking-water networks

Most naturally occurring biofilms contain a vast majority of microorganisms which have not yet been cultured, and therefore we have little information on the genetic information content of these communities. Therefore, we initiated work to characterize the complex metagenome of model drinking water biofilms grown on rubber-coated valves by employing three different strategies. First, a sequence analysis of 650 16S rRNA clones indicated a high diversity within the biofilm communities, with the majority of the microbes being closely related to the Proteobacteria: Only a small fraction of the 16S rRNA sequences were highly similar to rRNA sequences from Actinobacteria, low-G+C gram-positives and the Cytophaga-Flavobacterium-Bacteroides group. Our second strategy included a snapshot genome sequencing approach. Homology searches in public databases with 5,000 random sequence clones from a small insert library resulted in the identification of 2,200 putative protein-coding sequences, of which 1,026 could be classified into functional groups. Similarity analyses indicated that significant fractions of the genes and proteins identified were highly similar to known proteins observed in the genera Rhizobium, Pseudomonas, and Escherichia: Finally, we report 144 kb of DNA sequence information from four selected cosmid clones, of which two formed a 75-kb overlapping contig. The majority of the proteins identified by whole-cosmid sequencing probably originated from microbes closely related to the alpha-, beta-, and gamma-Proteobacteria: The sequence information was used to set up a database containing the phylogenetic and genomic information on this model microbial community. Concerning the potential health risk of the microbial community studied, no DNA or protein sequences directly linked to pathogenic traits were identified.     

A novel improved method for Aspergillus nidulans transformation

We systematically investigated the efficiency of Aspergillus nidulans transformation using protoplasts prepared from different stages of conidiospore germination and young mycelium. Using standard integrative plasmids, increased transformation yields were obtained with protoplasts isolated from a specific stage coincident with germ tube emergence. This increase ranged, on the average, from two- to eightfold depending on different plasmids used. Transformation efficiencies with a replicative plasmid were similar to those obtained using previously described methods. Although this observation suggests that elevated transformation efficiencies might be due to increased efficiency of recombination between plasmid and genomic sequences, we cannot exclude other factors associated with the particular developmental stage used. In the course of this study, we also examined the effect of other parameters that might enhance transformation yields. The method described is also significantly easier and faster than other current methods.