Species-crossreactive scFv against the tumor stroma marker "fibroblast activation protein" selected by phage display from an immunized FAP-/- knock-out mouse.

BACKGROUND: Fibroblast activation protein (FAP) is a type II membrane protein expressed on tumor stroma fibroblasts in more than 90% of all carcinomas. FAP serves as a diagnostic marker and is potential therapeutic target for treatment of a wide variety of FAP+ carcinomas. Murine tumor stroma models and FAP-specific antibodies are required to investigate the functional role of FAP in tumor biology and its usefulness for drug targeting. We here describe the development of antibodies with crossreactivity for human (hFAP) and murine FAP (mFAP), which share 89% amino acid identity. MATERIAL AND METHODS: An FAP-/- mouse was sequentially immunized with recombinant murine and human FAP-CD8 fusion proteins. Immunoglobulin cDNA derived from hyperimmune spleen cells was used for the construction of a combinatorial single chain Fv (scFv) library. Phage display selection of FAP-specific scFv was performed on immobilized hFAP followed by selection on cells expressing murine FAP. RESULTS: High-affinity, species-crossreactive, FAP-specific scFv were isolated upon sequential phage display selection. A bivalent derivative (minibody M036) constructed thereof was applied for immunohistochemical analyses and allowed detection of FAP expression on stroma cells of different human carcinomas as well as on murine host stroma in a tumor xenograft model. CONCLUSIONS: MB M036, derived from phage display selected species crossreactive scFv, is suitable for tumor stroma targeting and will be a valuable tool in the analyses of the functional role of FAP in tumor biology as well as in the evaluation of the suitability of FAP for drug targeting.     

Eicosanoids in insect immunity: bacterial infection stimulates hemocytic phospholipase A2 activity in tobacco hornworms

Intracellular phospholipase A(2) (PLA(2)) is responsible for releasing arachidonic acid from cellular phospholipids, and is thought to be the first step in eicosanoid biosynthesis. Intracellular PLA(2)s have been characterized in fat body and hemocytes from tobacco hornworms, Manduca sexta. Here we show that bacterial challenge stimulated increased PLA(2) activity in isolated hemocyte preparations, relative to control hemocyte preparations that were challenged with water. The increased activity was detected as early as 15 s post-challenge and lasted for at least 1 h. The increased activity depended on a minimum bacterial challenge dose, and was inhibited in reactions conducted in the presence of oleyoxyethylphosphorylcholine, a site-specific PLA(2) inhibitor. In independent experiments with serum prepared from whole hemolymph, we found no PLA(2) activity was secreted into serum during the first 24 h following bacterial infection. We infer that a hemocytic intracellular PLA(2) activity is increased immediately an infection is detected. The significance of this enzyme lies in its role in launching the biosynthesis of eicosanoids, which mediate cellular immune reactions to bacterial infection.     

Fate of intraluminal glutamine in the perfused renal tubule

To determine the fate of intraluminal glutamine and specifically the role of brush border gamma glutamyltransferase in its hydrolysis and reabsorption, proximal convoluted tubules of rabbits were isolated and perfused with an artificial ultrafiltrate containing 1 mM 14C-glutamine and 3H-PEG as a volume absorption marker. The tubules, average length 0.80 +/- 0.09 mm, were bathed in perfusate containing albumin, 6.5 percent but no glutamine. Aliquots of collectate and bathing media were monitored for total 14C counts while the distribution of radioactive 14C between glutamine and glutamate in the collectate was determined by separation on a Dowex X8 formate form ion-exchange column. After 3 ten minute control periods the perfusate was switched to one containing 1 mM AT-125 in addition to glutamine and after equilibration an additional 3 collections were obtained. Control period glutamine load averaged 16.1 +/- 2.4 pmole/min of which 35 percent was absorbed and 38 and 27 percent excreted as glutamine and glutamate respectively; of the absorbed glutamine 25 percent was metabolized. During AT-125 administration, glutamine delivery averaged 15.0 +/- 2.1 pmole/min of which 57 percent was absorbed; increased absorption occurred at the expence of intraluminal glutamate formation which fell to less than 10 percent. Thus luminal transport and gamma glutamyltransferase mediated hydrolysis appear to compete for available glutamine. Significantly, reducing intraluminal glutamine hydrolysis doubles the cellular metabolism of absorbed glutamine suggesting that extracellular conversion of glutamine to glutamate alters the metabolic fate of filtered glutamine.     

Draft Genome Sequence of Fusobacterium nucleatum subsp. fusiforme ATCC 51190T

Fusobacterium nucleatum, one of the major causative bacteria of periodontitis, is classified into five subspecies (nucleatum, polymorphum, vincentii, animalis, and fusiforme) on the basis of the several phenotypic characteristics and DNA homology. This is the first report of the draft genome sequence of F. nucleatum subsp. fusiforme ATCC 51190(T).     

Secretases as therapeutic targets for Alzheimer's disease

Accumulation of amyloid-β (Aβ) is widely accepted as the key instigator of Alzheimer’s disease (AD). The proposed mechanism is that accumulation of Aβ results in inflammatory responses, oxidative damages, neurofibrillary tangles and, subsequently, neuronal/synaptic dysfunction and neuronal loss. Given the critical role of Aβ in the disease process, the proteases that produce this peptide are obvious targets. The goal would be to develop drugs that can inhibit the activity of these targets. Protease inhibitors have proved very effective for treating other disorders such as AIDS and hypertension. Mutations in APP (amyloid-β precursor protein), which flanks the Aβ sequence, cause early-onset familial AD, and evidence has pointed to the APP-to-Aβ conversion as a possible therapeutic target. Therapies aimed at modifying Aβ-related processes aim higher up the cascade and are therefore more likely to be able to alter the progression of the disease. However, it is not yet fully known whether the increases in Aβ levels are merely a result of earlier events that were already causing the disease.     

Substrate-binding loop interactions with pseudouridine trigger conformational changes that promote catalytic efficiency of pseudouridine kinase PUKI

Pseudouridine, one major RNA modification, is catabolized into uracil and ribose-5′-phosphate by two sequential enzymatic reactions. In the first step, pseudouridine kinase (PUKI) phosphorylates pseudouridine to pseudouridine 5′-monophosphate. High-fidelity catalysis of pseudouridine by PUKI prevents possible disturbance of in vivo pyrimidine homeostasis. However, the molecular basis of how PUKI selectively phosphorylates pseudouridine over uridine with >100-fold greater efficiency despite minor differences in their K_m values has not been elucidated. To investigate this selectivity, in this study we determined the structures of PUKI from Escherichia coli strain B (EcPUKI) in various ligation states. The structure of EcPUKI was determined to be similar to PUKI from Arabidopsis thaliana, including an α/β core domain and β-stranded small domain, with dimerization occurring via the β-stranded small domain. In a binary complex, we show that Ser30 in the substrate-binding loop of the small domain mediates interactions with the hallmark N1 atom of pseudouridine nucleobase, causing conformational changes in its quaternary structure. Kinetic and fluorescence spectroscopic analyses also showed that the Ser30-mediated interaction is a prerequisite for conformational changes and subsequent catalysis by EcPUKI. Furthermore, S30A mutation or EcPUKI complexed with other nucleosides homologous to pseudouridine but lacking the pseudouridine-specific N1 atom did not induce such conformational changes, demonstrating the catalytic significance of the proposed Ser30-mediated interaction. These analyses provide structural and functional evidence for a pseudouridine-dependent conformational change of EcPUKI and its functional linkage to catalysis.     

Pitaya (stenocereus spp., Cactaceae): An ancient and modern fruit crop of Mexico

Pitayasfrom various species were an important edible fruit in semiarid lands of tropical and subtropical Mexico in ancient times. Recently, farmers have been cultivating plants selected from the wild, such as Stenocereus queretaroensis in the Sayula Basin of Jalisco. These cacti can flower and produce fruit before the onset of the summer rainy period. Their fruits have an attractively colored pulp (often dark red) with digestible seeds and without the nasty glochids found on cactus pears. The sugar content is 10 to 11%. The shelf life is only a few days, as the fruits tend to dehisce longitudinally. Pitayas bring a competitive price in local markets, resulting in a substantial financial return with relatively low inputs of water, fertilizer, and pesticides.     

The complete mitochondrial genome of human parasitic roundworm, Ascaris lumbricoides

The genome length of the Ascaris lumbricoides, human parasitic roundworm, is 14,281 bp with a nucleotide composition of 22.1% A, 49.8% T, 7.8% C, and 20.3% G. The genome consists of 12 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 control region.