Using weighted fixed neural networks for unsupervised fuzzy clustering

A novel algorithm for unsupervised fuzzy clustering is introduced. The algorithm uses a so-called Weighted Fixed Neural Network (WFNN) to store important and useful information about the topological relations in a given data set. The algorithm produces a weighted connected net, of weighted nodes connected by weighted edges, which reflects and preserves the topology of the input data set. The weights of the nodes and the edges in the resulting net are proportional to the local densities of data samples in input space. The connectedness of the net can be changed, and the higher the connectedness of the net is chosen, the fuzzier the system becomes. The new algorithm is computationally efficient when compared to other existing methods for clustering multi-dimensional data, such as color images.     

Pathophysiological contributions of fucosyltransferases in renal ischemia reperfusion injury

Ischemia reperfusion injury (IRI) is a major cause of delayed graft function. Recent studies have shown that selectins play an important role in IRI. Selectins bind to sialylated and fucosylated sLe(x) receptors, and two enzymes, fucosyltransferase IV (FucT-IV) and VII (FucT-VII), are important in the function of these receptors. We hypothesized that fucosyltransferase (FucT) enzymes were important pathophysiologic mediators of renal IRI. We therefore evaluated renal IRI in mice deficient in FucT-IV, FucT-VII, and both FucT-IV and FucT-VII and compared their renal function, tubular injury, selectin ligand expression, and neutrophil infiltration to those in wild-type control mice. Bilateral 30-min renal IRI was performed, and the results demonstrated that mice deficient in both FucT-IV/FucT-VII were significantly protected from renal IRI at 24 and 48 h compared with wild-type control mice. FucT-IV-deficient mice showed only modest protection from renal injury at 24 h. However, FucT-VII-deficient mice had similar injury as wild-type mice. Histological analysis of kidney tissue postischemia revealed that mice deficient in both FucT-IV and FucT-VII had significantly reduced tubular injury compared with wild-type mice. Selectin ligand expression increased postischemia in wild-type, but not FucT-IV/FucT-VII-deficient, mice. Neutrophil infiltration in postischemic kidneys of FucT-IV/FucT-VII-deficient mice was also attenuated. These data demonstrate that fucosyltransferases are important in the pathogenesis of renal IRI and are potential therapeutic targets.     

Impact of endogenous nitric oxide on microglial cell energy metabolism and labile iron pool

Microglial activation is common in several neurodegenerative disorders. In the present study, we used the murine BV-2 microglial cell line stimulated with gamma-interferon and lipopolysaccharide to gain new insights into the effects of endogenously produced NO on mitochondrial respiratory capacity, iron regulatory protein activity, and redox-active iron level. Using polarographic measurement of respiration of both intact and digitonin-permeabilized cells, and spectrophotometric determination of individual respiratory chain complex activity, we showed that in addition to the reversible inhibition of cytochrome-c oxidase, long-term endogenous NO production reduced complex-I and complex-II activities in an irreversible manner. As a consequence, the cellular ATP level was decreased in NO-producing cells, whereas ATPase activity was unaffected. We show that NO up-regulates RNA-binding of iron regulatory protein 1 in microglial cells, and strongly reduces the labile iron pool. Together these results point to a contribution of NO derived from inflammatory microglia to the misregulation of energy-producing reactions and iron metabolism, often associated with the pathogenesis of neurodegenerative disorders.     

Crystal structure of the BEACH domain reveals an unusual fold and extensive association with a novel PH domain

The BEACH domain is highly conserved in a large family of eukaryotic proteins, and is crucial for their functions in vesicle trafficking, membrane dynamics and receptor signaling. However, it does not share any sequence homology with other proteins. Here we report the crystal structure at 2.9 A resolution of the BEACH domain of human neurobeachin. It shows that the BEACH domain has a new and unusual polypeptide backbone fold, as the peptide segments in its core do not assume regular secondary structures. Unexpectedly, the structure also reveals that the BEACH domain is in extensive association with a novel, weakly conserved pleckstrin-homology (PH) domain. Consistent with the structural analysis, biochemical studies show that the PH and BEACH domains have strong interactions, suggesting they may function as a single unit. Functional studies in intact cells demonstrate the requirement of both the PH and the BEACH domains for activity. A prominent groove at the interface between the two domains may be used to recruit their binding partners.     

Differential sensitivity of oleosins to proteolysis during oil body mobilization in sunflower seedlings

Until now, there has been no conclusive demonstration of any in vivo oleosin degradation at the early stages of oil body mobilization. The present work on sunflower (Helianthus annuus L.) has demonstrated limited oleosin degradation during seed germination. Seedling cotyledon homogenization in Tris-urea buffer, followed by SDS-PAGE, revealed three oleosins (16, 17.5 and 20 kDa). Incubation of oil bodies with total soluble protein from 4-day-old seedlings resulted in oleosin degradation. In vitro and in vivo degradation of the 17.5-kDa oleosin was faster than the other two, indicating its greater susceptibility to proteolysis. Oleosin degradation by the total soluble protein resulted in a transient 14.5-kDa polypeptide, followed by an 11-kDa protease-protected fragment, which appeared post-germinatively and accumulated corresponding to increased rate of lipid mobilization. A 65-kDa protease, active at pH 7.5-9.5, was zymographically detected in the total soluble protein. Its activity increased along with in vivo accumulation of the protease-protected fragment during seed germination and accompanying lipid mobilization. Protease-treated oil bodies were more susceptible to maize lipase action. Differential proteolytic sensitivity of different oleosins in the oil body membranes could be a determinant of oil body longevity during seed germination.     

Characterization of Cah,a calcium-binding and heat-extractable autotransporter protein of enterohaemorrhagic Escherichia coli

We have identified and characterized a protein of enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7 that shares homology with antigen 43 and AIDA-I of E. coli. The gene encoding this protein consists of a 2850 bp open reading frame and was named cah for calcium binding antigen 43 homologue. The prototype EHEC strain EDL933 possesses identical duplicate copies of cah (cah1 and cah2), which showed 100% identity at the nucleotide level. We showed that E. coli K-12 containing the recombinant cah gene produced two proteins, an approximately 80 kDa outer membrane protein and a 43.0 kDa heat-extractable protein. The Cah protein contains a predicted 52-amino-acid extended signal sequence found in several autotransporter proteins, and N-terminal sequencing data indicated that the 43.0 kDa passenger protein was derived from cleavage of the signal sequence from alanine at position 53. Phenotypes such as autoaggregation and change in bacterial shape were observed when a recombinant plasmid containing the cah gene was introduced into a laboratory E. coli strain, and these phenotypes were eliminated upon mutation of the cah gene. The passenger domain contains six domains found in calcium-binding proteins, and the recombinant Cah passenger protein bound 45Ca2+. In E. coli O157:H7, Cah is a heat-extractable protein, the expression of which is induced in minimal essential media and under divalent ion-depleting conditions; it also participates in the formation of biofilms. Our results provide insight into the expression, secretion and preliminary features of the calcium-binding Cah autotransporter protein of EHEC O157:H7.     

Periodic change in DO concentration for efficient poly-β-hydroxy-butyrate production using temperature-inducible recombinant<Emphasis Type="Italic">Escherichia coli</Emphasis> with proteome analysis

RecombinantEscherichia coli strain harboring the λp _R-p _L promotor and heterologus poly-β-hydroxybutyrate (PHB) biosynthesis genes was used to investigate the effect of culture conditions on the efficient PHB production. The expression ofphb genes was induced by a temperature upshift from 33°C to 38°C. The protein expression levels were measured by using two-dimensional electrophoresis, and the enzyme activities were also measured to understand the effect of culture temperature, carbon sources, and the dissolved oxygen (DO) concentration on the metabolic regulations. AcetylCoA is an important branch point for PHB production. The decrease in DO concentration lowers the citrate synthase activity, thus limit the flux toward the TCA cycle, and increase the flux for PHB production. Since NADPH is required for PHB production, the PHB production does not continue leading the overproduction of acetate and lactate. Based on these observations, a new operation was considered where DO concentration was changed periodically, and it was verified its usefulness for the efficient PHB production by experiments.     

Genetic variation of Trigonobalanus verticillata,a primitive species of Fagaceae,in Malaysia revealed by chloroplast sequences and AFLP markers

The genetic variation of Trigonobalanus verticillata, the most recently described genus of Fagaceae, was studied using chloroplast DNA sequences and AFLP fingerprinting. This species has a restricted distribution that is known to include seven localities in tropical lower montane forests in Malaysia and Indonesia. A total of 75 individuals were collected from Bario, Kinabalu, and Fraser's Hill in Malaysia. The sequences of rbcL, matK, and three non-coding regions (atpB-rbcL spacer, trnL intron, and trnL-trnF spacer) were determined for 19 individuals from these populations. We found a total of 30 nucleotide substitutions and four length variations, which allowed identification of three haplotypes characterizing each population. No substitutions were detected within populations, while the tandem repeats in the trnL -trnF spacer had a variable repeat number of a 20-bp motif only in Kinabalu. The differentiation of the populations inferred from the cpDNA molecular clock calibrated with paleontological data was estimated to be 8.3 MYA between Bario and Kinabalu, and 16.7 MYA between Fraser's Hill and the other populations. In AFLP analysis, four selective primer pairs yielded a total of 431 loci, of which 340 (78.9%) were polymorphic. The results showed relatively high gene diversity (H(S) = 0.153 and H(T) = 0.198) and nucleotide diversity (pi(S) = 0.0132 and pi(T) = 0.0168) both within and among the populations. Although the cpDNA data suggest that little or no gene flow occurred between the populations via seeds, the fixation index estimated from AFLP data (F(ST) = 0.153 and N(ST) = 0.214) implies that some gene flow occurs between populations, possibly through pollen transfer.