Single chain antibody fragments for ocular use produced at high levels in a commercial wheat variety

We are investigating the use of single chain antibody fragments (scFv) in eye drops for diagnosis and treatment of eye diseases. For ocular use, recombinant proteins must be free of bacterial endotoxin that causes inflammation in the eye. We required a means of generating high yields of scFvs with little endotoxin contamination. Using microprojectile bombardment we produced transgenic lines of the commercial wheat variety, Westonia, that express two scFvs that bind to CD4 or CD28 on the surface of rat thymocytes. A high level of expression of active scFv in the range 50-180 microg/g was measured by quantitative flow cytometry in crude extracts made from mature seeds. The levels of expression were stable over four generations of transgenic plants and mature seeds were stored for one year with little loss of scFv activity. Substantial purification of scFv was achieved by immobilised metal affinity chromatography. Compared to bacterial extracts, crude transgenic seed extracts contained only a small amount of endotoxin (150 EU/ml) that will be easily removed by purification. The transgenic wheat lines express functional scFv at levels comparable to production in bacteria and promise to be superior to bacteria for production of scFv pharmaceuticals for ocular use.     

His164 regulates accessibility to the active site in fungal 17beta-hydroxysteroid dehydrogenase

17beta-Hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is an NADPH-dependent member of the short-chain dehydrogenase/ reductase superfamily. To study the catalytic properties of this enzyme, we prepared several specific mutations of 17beta-HSDcl (Tyr167Phe, His164Trp/Gly, Tyr212Ala). Wild-type 17beta-HSDcl and the 17beta-HSDcl mutants were evaluated by chromatographic, kinetic and thermodynamic means. The Tyr167Phe mutation resulted in a complete loss of enzyme activity, while substitution of His164 with Trp and Gly both resulted in higher specificity number (V/K) for the steroid substrates, which are mainly a consequence of easier accessibility of steroid substrates to the active-site hollow under optimized conditions. The Tyr212Ala mutant showed increased activity in the oxidative direction, which appears to be a consequence of increased NADPH dissociation. The kinetic characterizations and thermodynamic analyses also suggest that His164 and Tyr212 in 17beta-HSDcl have a role in the opening and closing of the active site of this enzyme and in the discrimination between oxidized and reduced coenzyme.     

Characterization and biological role of the O-polysaccharide gene cluster of Yersinia enterocolitica serotype O:9

Yersinia enterocolitica serotype O:9 is a gram-negative enteropathogen that infects animals and humans. The role of lipopolysaccharide (LPS) in Y. enterocolitica O:9 pathogenesis, however, remains unclear. The O:9 LPS consists of lipid A to which is linked the inner core oligosaccharide, serving as an attachment site for both the outer core (OC) hexasaccharide and the O-polysaccharide (OPS; a homopolymer of N-formylperosamine). In this work, we cloned the OPS gene cluster of O:9 and identified 12 genes organized into four operons upstream of the gnd gene. Ten genes were predicted to encode glycosyltransferases, the ATP-binding cassette polysaccharide translocators, or enzymes required for the biosynthesis of GDP-N-formylperosamine. The two remaining genes within the OPS gene cluster, galF and galU, were not ascribed a clear function in OPS biosynthesis; however, the latter gene appeared to be essential for O:9. The biological functions of O:9 OPS and OC were studied using isogenic mutants lacking one or both of these LPS parts. We showed that OPS and OC confer resistance to human complement and polymyxin B; the OPS effect on polymyxin B resistance could be observed only in the absence of OC.     

Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP)

Rab GTPases recruit effector proteins, via their GTP-dependent switch regions, to distinct subcellular compartments. Rab11 and Rab25 are closely related small GTPases that bind to common effectors termed the Rab11 family of interacting proteins (FIPs). The FIPs are organized into two subclasses (class I and class II) based on sequence and domain organization, and both subclasses contain a highly conserved Rab-binding domain at their C termini. Yeast two-hybrid and biochemical studies have revealed that the more distantly related Rab14 also interacts with class I FIPs. Here, we perform detailed structural, thermodynamic, and cellular analyses of the interactions between Rab14 and one of the class I FIPs, the Rab-coupling protein (RCP), to clarify the molecular aspects of the interaction. We find that Rab14 indeed binds to RCP, albeit with reduced affinity relative to conventional Rab11-FIP and Rab25-FIP complexes. However, in vivo, Rab11 recruits RCP onto biological membranes. Furthermore, biophysical analyses reveal a noncanonical 1:2 stoichiometry between Rab14-RCP in dilute solutions, in contrast to Rab11/25 complexes. The structure of Rab14-RCP reveals that Rab14 interacts with the canonical Rab-binding domain and also provides insight into the unusual properties of the complex. Finally, we show that both the Rab coupling protein and Rab14 function in neuritogenesis.     

Molecular markers reconstruct the invasion history of variable leaf watermilfoil (<Emphasis Type="Italic">Myriophyllum heterophyllum</Emphasis>) and distinguish it from closely related species

Genetic variation is increasingly recognized as an important factor influencing the establishment and spread of introduced species, and depends on both the introduction history and partitioning of genetic variation within and among potential source populations. We examine patterns of genetic variation in native and introduced populations of variable leaf watermilfoil, Myriophyllum heterophyllum, using chloroplast (trnL-F) and ribosomal (ITS) DNA sequences, as well as amplified fragment length polymorphisms (AFLPs). We identify a strong phylogeographic break distinguishing populations located on the Atlantic Coastal Plain (ACP) versus other (“Continental”) portions of the native range. Within these distinct biogeographic regions, we also find genetic variation to be strongly partitioned among populations as analysis of molecular variance (AMOVA) partitioned 91 and 75% of cpDNA and ITS diversity among populations, respectively. We demonstrate that the introduced ranges of variable leaf watermilfoil (northeastern and western US) result from multiple independent introductions from a variety of source populations, including lineages from both the ACP and Continental portions of the native range. In addition, we used our molecular markers to demonstrate that variable leaf watermilfoil is genetically distinct from three closely-related species that it is morphologically similar to. In particular, we demonstrate that M. heterophyllum is clearly distinct from a morphologically similar native species in the western US, M. hippuroides—whose distinctiveness from M. heterophyllum has been questioned—and therefore confirm the introduction of M. heterophyllum in the western US. Furthermore, we provide the first evidence for hybridization between these two species. Finally, our molecular markers identify previously unrecognized genetic variation in these four species, and therefore demonstrate the need for further taxonomic investigation.     

Phytoestrogens as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase

Different phytoestrogens were tested as inhibitors of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a member of the short-chain dehydrogenase/reductase superfamily. Phytoestrogens inhibited the oxidation of 100microM 17beta-hydroxyestra-4-en-3-one and the reduction of 100microM estra-4-en-3,17-dione, the best substrate pair known. The best inhibitors of oxidation, with IC(50) below 1microM, were flavones hydroxylated at positions 3, 5 and 7: 3-hydroxyflavone, 3,7-dihydroxyflavone, 5,7-dihydroxyflavone (chrysin) and 5-hydroxyflavone, together with 5-methoxyflavone. The best inhibitors of reduction were less potent; 3-hydroxyflavone, 5-methoxyflavone, coumestrol, 3,5,7,4'-tetrahydroxyflavone (kaempferol) and 5-hydroxyflavone, all had IC(50) values between 1 and 5microM. Docking the representative inhibitors chrysin and kaempferol into the active site of 17beta-HSDcl revealed the possible binding mode, in which they are sandwiched between the nicotinamide moiety and Tyr212. The structural features of phytoestrogens, inhibitors of both oxidation and reduction catalyzed by the fungal 17beta-HSD, are similar to the reported structural features of phytoestrogen inhibitors of human 17beta-HSD types 1 and 2.     

Yeast growth selection system for detecting activity and inhibition of dimerization-dependent receptor tyrosine kinase

Receptor tyrosine kinases (RTKs) play an important role in the control of fundamental cellular processes, including cell proliferation, migration, differentiation, and survival. Deregulated RTK signaling is critically involved in the development and progression of human cancer. Here, we present an assay for monitoring RTK activities in yeast, which provides an ideal heterologous cellular system to study these mammalian proteins in a null background environment. With our system, we have reconstituted aspects of the epidermal growth factor receptor (EGFR) signaling pathway as a model. Our approach is based on the Ras-recruitment system, in which membrane localization of a constitutively active human Ras achieved through protein-protein interactions can rescue growth of a temperature-sensitive yeast strain (cdc25-2). We show that co-expression of a dimerizing membrane-bound EGFR variant with specific adaptor proteins fused to the active Ras rescues growth of the cdc25-2 mutant yeast strain at the nonpermissive temperature. Using kinase-defective RTK mutants and selective EGFR kinase inhibitors, we demonstrate that growth rate of this yeast strain correlates with kinase activity of the EGFR derivatives. The RTK cellular assay presented here can be applied in high-throughput screens for selecting RTK-specific inhibitors that must be able to permeate the membrane and to function in an eukaryotic intrecellular environment.     

Nonsteroidal anti-inflammatory drugs and their analogues as inhibitors of aldo-keto reductase AKR1C3: new lead compounds for the development of anticancer agents

Nonsteroidal anti-inflammatory drugs (NSAIDs) like indomethacin, flufenamic acid, and related compounds have been recently identified as potent inhibitors of AKR1C3. We report that some other NSAIDs (diclofenac and naproxen) also inhibit AKR1C3, with the IC(50) values in the low micromolar range. In order to obtain more information about the structure-activity relationship and to identify new leads, a series of compounds designed on the basis of NSAIDs were synthesized and screened on AKR1C3. The most active compounds were 2-[(2,2-diphenylacetyl)amino]benzoic acid 4 (IC(50)=11microM) and 3-phenoxybenzoic acid 10 (IC(50)=0.68microM). These compounds represent promising starting points for the development of new anticancer agents.     

Amplification of KIT, PDGFRA, VEGFR2, and EGFR in gliomas

Receptor tyrosine kinase aberrations are implicated in the genesis of gliomas. We investigated expression and amplification of KIT, PDGFRA, VEGFR2, and EGFR in 87 gliomas consisting of astrocytomas, anaplastic astrocytomas, oligodendrogliomas, or oligoastrocytomas in tumor samples collected at the time of the diagnosis and in samples of the same tumors at tumor recurrence. Gene amplifications were investigated using either chromogenic in situ hybridization or fluorescence in situ hybridization, and protein expression using immunohistochemistry. In samples collected at glioma diagnosis, KIT and PDGFRA amplifications were more frequent in anaplastic astrocytomas than in astrocytomas, oligodendrogliomas, and oligoastrocytomas [28% versus 5% (P = 0.012) and 33% versus 2% (P = 0.0008), respectively]. VEGFR2 amplifications occurred in 6% to 17% of the gliomas at diagnosis, and EGFR amplifications in 0% to 12%. Amplified KIT was more frequently present in recurrent gliomas than in newly diagnosed gliomas (P = 0.0066). KIT amplification was associated with KIT protein expression and with presence of PDGFRA and EGFR amplifications both at the time of the first glioma diagnosis and at tumor recurrence, and with VEGFR2 amplification at tumor recurrence. Three (4%) primary gliomas and 10 (14%) recurrent gliomas that were evaluable for coamplification of KIT, PDGFRA, and VEGFR2 showed amplification of at least two of these genes; the amplicon contained amplified KIT in all 13 cases. In conclusion, besides glioblastoma, amplified KIT, PDGFRA, and VEGFR may also occur in lower-grade gliomas and in their recurrent tumors. It is currently not known whether specific tyrosine kinase inhibitors are effective in the treatment of such gliomas.     

The circulatory influence on development of age-related macular degeneration and hearing and equilibrium impairments

This study attempts to answer the question if any level of head and neck circulation takes a part in development of Age-Related Macular Degeneration (ARMD) and hearing and equilibrium impairments. Condition of large blood vessels was examined by Color-Doppler ultrasound, and carotid and ophthalmic arteries were included. The microcirculatory changes were examined directly by fundus photography and fluorescein angiography and indirectly testing hearing and equilibrium. The study group included 40 patients (21 females, 19 males) aging from 53 to 84 years with different stages of ARMD. The control group included 40 patients (18 females, 22 males) aging from 51 to 82 years without ARMD. Patients were inhabitants of Primorsko-Goranska County. There was no relationship between ARMD and condition of large blood vessels because significant stenosis of carotid arteries was found in 2 patients (5%) in study group and 3 patients (7.5%) in the control group (p > 0.05). On the contrary, we found correlation between ARMD and hearing (p = 0.0127) and equilibrium impairments (p = 0.0242). Fluorescein angiograms shows raised number of ischemic retinal capillaries in patients with ARMD (p = 0.0053). Results lead to conclusion that circulatory disorders on microcirculatory level take a great part in development of ARMD and hearing and equilibrium impairments in the elderly. The key is damage of sensory cells of the retina and inner ear caused by microcirculatory disorders. Interesting data was noticed that 9 patients with more serious ARMD on one side of head had greater hearing loss on the same side. If we find a new treatment for microcirculatory disorders, maybe we can treat both sensory impairments in earlier stage.