Comparison of Cobb Angles Measured Manually,Calculated from 3-D Spinal Reconstruction,and Estimated from Torso Asymmetry

While scoliotic spinal deformity is traditionally measured by the Cobb angle, we seek to estimate scoliosis severity from the torso surface without X-ray radiation. Here, we measured the Cobb angle in three ways: by protractor from postero-anterior X-ray, by computer from a 3-D digitized model of the vertebral body line, and by neural-network estimation from indices of torso surface asymmetry. The estimates of the Cobb angle by computer and by neural network were equally accurate in 153 records from 52 patients (standard deviation of 6° from the Cobb angle, r =0.93 ), showing that torso asymmetry reliably predicted spinal deformity. Further improvements in predictive accuracy may require estimation of other 3-D indices of spinal deformity besides the Cobb angle with its wide measurement variability.     

Importance of mitochondrial dynamics during meiosis and sporulation

Opposing fission and fusion events maintain the yeast mitochondrial network. Six proteins regulate these membrane dynamics during mitotic growth-Dnm1p, Mdv1p, and Fis1p mediate fission; Fzo1p, Mgm1p, and Ugo1p mediate fusion. Previous studies established that mitochondria fragment and rejoin at distinct stages during meiosis and sporulation, suggesting that mitochondrial fission and fusion are required during this process. Here we report that strains defective for mitochondrial fission alone, or both fission and fusion, complete meiosis and sporulation. However, visualization of mitochondria in sporulating cultures reveals morphological defects associated with the loss of fusion and/or fission proteins. Specifically, mitochondria collapse to one side of the cell and fail to fragment during presporulation. In addition, mitochondria are not inherited equally by newly formed spores, and mitochondrial DNA nucleoid segregation defects give rise to spores lacking nucleoids. This nucleoid inheritance defect is correlated with an increase in petite spore colonies. Unexpectedly, mitochondria fragment in mature tetrads lacking fission proteins. The latter finding suggests either that novel fission machinery operates during sporulation or that mechanical forces generate the mitochondrial fragments observed in mature spores. These results provide evidence of fitness defects caused by fission mutations and reveal new phenotypes associated with fission and fusion mutations.     

Methylation by a Unique α-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725

Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the Gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute) and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation.     

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H₂O₂), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H₂O₂ in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.Download video file.^{(79M, mov)}     

Expression of transglutaminase substrate activity on Candida albicans germ tubes through a coiled, disulfide-bonded N-terminal domain of Hwp1 requires C-terminal glycosylphosphatidylinositol modification

By serving as a microbial substrate for epithelial cell transglutaminase, Hwp1 (Hyphal wall protein 1) of Candida albicans participates in cross-links with proteins on the mammalian mucosa. Biophysical properties of the transglutaminase substrate domain were explored using a recombinant protein representative of the N-terminal domain of Hwp1 and were similar to other transglutaminase substrates, the small proline-rich proteins of cornified envelopes found in stratified squamous epithelia. Recombinant Hwp1 lacks alpha and beta structures by circular dichroism and likely exists as a disulfide-cross-linked coiled-coil. The transglutaminase substrate property prompted a unique approach for investigating the features of surface Hwp1 on germ tubes. A lysine analog, 5-(biotinamido)pentylamine, was cross-linked to germ tubes catalyzed by transglutaminase 2 prior to cell fractionation, immunoprecipitation, and detection with streptavidin conjugates. The majority of the transglutaminase-modifiable Hwp1 was covalently attached to the beta-glucan of hyphae by the C terminus of Hwp1 via a glycosylphosphatidylinositol remnant anchor. A putative precursor of cell wall forms of Hwp1 was identified in the cell extract and in the culture medium. Hwp1 was modified by relatively short N-linked glycans, and the molecular size of the protein was reduced by hypomannosylation when expressed in O-glycosylation mutant strains. Hwp1 combines features of mammalian transglutaminase substrate proteins with characteristics of fungal cell wall proteins to form an unconventional adhesin at the hyphal wall of C. albicans.     

Characterization of the Lipid Linkage Region and Chain Length of the Cellubiuronic Acid Capsule of Streptococcus pneumoniae

The processive reaction mechanisms of β-glycosyl-polymerases are poorly understood. The cellubiuronan synthase of Streptococcus pneumoniae catalyzes the synthesis of the type 3 capsular polysaccharide through the alternate additions of β-1,3-Glc and β-1,4-GlcUA. The processive multistep reaction involves the sequential binding of two nucleotide sugar donors in coordination with the extension of a polysaccharide chain associated with the carbohydrate acceptor recognition site. Degradation analysis using cellubiuronan-specific depolymerase demonstrated that the oligosaccharide-lipid and polysaccharide-lipid products synthesized in vitro with recombinant cellubiuronan synthase had a similar oligosaccharyl-lipid at their reducing termini, providing definitive evidence for a precursor-product relationship and also confirming that growth occurred at the nonreducing end following initiation on phosphatidylglycerol. The presence of a lipid marker at the reducing end allowed the quantitative determination of cellubiuronic acid polysaccharide chain lengths. As the UDP-GlcUA concentration was increased from 1 to 11.5 μm, the level of synthase in the transitory processive state decreased, with the predominant oligosaccharide-lipid product containing 3 uronic acid residues, whereas the proportion of synthase in the fully processive state increased and the polysaccharide chain length increased from 320 to 6700 monosaccharide units. In conjunction with other kinetic data, these results suggest that the formation of a complex between a tetrauronosyl oligomer and the carbohydrate acceptor recognition site plays a central role in coordinating the repetitive interaction of the synthase with the nucleotide sugar donors and modulating the chain length of cellubiuronan polysaccharide.Cellubiuronic acid, the capsular polysaccharide of type 3 Streptococcus pneumoniae, is composed of the repeating disaccharide cellobiuronic acid (-3)-β-d-GlcUA²-(1,4)-β-d-Glc-(1-) (1) and is synthesized by a processive mechanism similar to that for cellulose, chitin, hyaluronic acid, and other related β-glycans (2). This group of polysaccharides is synthesized by inverting GT-2A polymerases that are located in the plasma membrane with their active sites on the cytoplasmic face, and following chain initiation, the synthases are thought to be involved in the extrusion of the nascent chains to the external membrane face (3–8). The overall processive elaboration of these polysaccharides remains poorly understood at the molecular level. In particular, there is relatively little information concerning the initiation process, the facilitation of chain extrusion, the mechanism of translocation, and the regulation of the final chain length during the assembly of these polymers. Recent investigations in this laboratory have begun to unravel some of the details of both the early and later stages of the biosynthesis of cellubiuronan.Unlike most S. pneumoniae capsules, whose elaboration requires multiple glycosyltransferases, a polymerase, and an additional transport system (9), the assembly and transport of cellubiuronic acid in type 3 strains is carried out by the single enzyme cellubiuronan synthase (Cps3S) (3, 10, 11). Studies of the synthase in S. pneumoniae and recombinant Escherichia coli membranes have shown that assembly of the polysaccharide involves two distinct kinetic phases: 1) a transitory processive state wherein the chain is thought to be initiated by the formation of an oligosaccharide-lipid that is loosely associated with the synthase, and 2) a fully processive state in which the polysaccharide is tightly bound to the carbohydrate substrate recognition site, except for a brief period during the translocation stage of each catalytic cycle (5, 12). Each catalytic cycle in the extrusion mode provides for chain extension by the addition of a repeating disaccharide and requires the alternate association of the synthase with UDP-Glc and UDP-GlcUA, the formation of the glycosidic linkages of the respective sugars, and the release, translocation, and reattachment of the elongating chain at the synthase carbohydrate recognition site. Transition from the transitory mode to the fully processive extrusion mode correlates with the attainment of a threshold-length oligosaccharide of ∼8 sugars (12). Nod factor chito-oligosaccharides from rhizobia are synthesized by a related group of synthases that apparently are not capable of organizing into an extrusion mode (13). Significantly, the maximum length of any reported Nod-factor oligosaccharide is 6 sugars (14).Based on β-glucosidase sensitivity of singly added [¹⁴C]Glc to the terminal end of high molecular weight cellubiuronan, it was deduced that the polysaccharide grows by repetitive β-1,3-Glc and β-1,4-GlcUA additions to the nonreducing terminus (2). Oligosaccharide-lipid assembly is thought to initiate on phosphatidylglycerol (15). To date, however, there has been no quantitative demonstration of polysaccharide-lipid conjugate, and the similarity of the distal sugar-lipid linkages in the polysaccharide- and oligosaccharide-lipid products has not been verified.Cellubiuronan depolymerase is a Bacillus circulans β-endoglucuronidase that specifically cleaves cellubiuronic acid chains at GlcUA-β1,4-Glc linkages (16, 17). The polysaccharide is successively cleaved at random internal linkages, which upon completion of hydrolysis yields a series of oligosaccharide end products containing 1–4 Glc-β1,3-GlcUA disaccharide units, with the most abundant oligomer being a tetrasaccharide. The high degree of specificity of this depolymerase has provided a sensitive analytical tool to further characterize cellubiuronan oligosaccharide- and polysaccharide-lipids, and even more importantly, it has provided a means for determining the chain length of these polysaccharides. Both in vivo (18) and in vitro studies (12) indicate that the processivity of cellubiuronan synthase is modulated by the concentration of UDP-GlcUA. However, lacking well defined cellubiuronan polysaccharide size standards, there has been no way to clearly establish the actual length of the polymer synthesized under different reaction conditions. The methodology described in this article has allowed a clear demonstration of the relationship between the polysaccharide size and the UDP-GlcUA substrate concentration and in turn has led to the development of a kinetic model (38), which provides both for UDP-sugar modulation of polysaccharide chain length and for the assembly by a single-site synthase of a polysaccharide composed of a repeating heterodisaccharide.     

Isolation and sequence analysis of P450 genes from a pyrethroid resistant colony of the major malaria vector Anopheles funestus.

Pyrethroid resistance has been demonstrated in populations of Anopheles funestus from South Africa and southern Mozambique. Resistance is associated with elevated P450 monooxygenase enzymes. In this study, degenerate primers based on conserved regions of Anopheles gambiae P450 CYP4, 6 and 9 families were used to amplify genomic and cDNA templates from A. funestus. A total of 12 CYP4, 12 CYP6 and 7 CYP9 partial genes have been isolated and sequenced. BLAST results revealed that A. funestus P450s generally have a high sequence identity to A. gambiae with above 75% identity at the amino acid level. The exception is CYP9J14. The A. gambiae P450 showing highest identity to CYP9J14 exhibits only 55% identity suggesting that CYP9J14 may have arisen from a recent duplication event. Molecular phylogenetic analysis based on amino acid sequences also supported this hypothesis. Intron positions, but not size, were highly conserved between the two species. The high level of orthology that exists in the P450 gene families of these two species may facilitate the prediction of individual P450 protein function.     

Reluctant Muslims: embodied hegemony and moral resistance in a Giriama spirit possession complex

This account of a form of spirit possession widely experienced among Giriama people of coastal Kenya challenges prevailing theories of possession as resistance. Giriama are routinely possessed by Muslim spirits which hold their bodies hostage, afflicting them with illness and vomiting until they agree to abandon their customary practices and embrace Islam. Those possessed apparently somatize a hegemonic system of oppressive meanings according to which Giriama ethnicity is essentially different from, and polluting to, Islam. Yet the same individuals who embody hegemony in this way may reflect upon their possession experience by articulating a defiant ideology of resistance against both the possessing spirit and the Muslim ethnic groups that the spirit represents. These observations thus highlight the complexity of the relationship between hegemony and the individual; they also provide a reminder that the idiom of possession does not necessarily articulate with power structures in a predictable and straightforward fashion.