Partial characterization of chorionic gonadotropin-like binding sites from the bacteria Xanthomonas maltophilia

The gram-negative bacterium, Xanthomonas maltophilia, has low- and high-affinity luteinizing hormone/chorionic gonadotropin (LH/CG)-binding sites, similar to the LH/CG receptor found in mammals. Although the low-affinity site binds both LH and human CG (hCG), the high-affinity site is specific for hCG. In the current investigation, these two binding sites were independently isolated from X. maltophilia for further characterization. To isolate functional binding sites, we developed a solubilization method using the detergent zwittergent 3,14 and high glycerol concentrations that allowed for the maintenance of ligand-binding integrity. Gel filtration experiments established molecular weights of 170 and 11.5 kDa for the two binding sites, which were supported by data from photoaffinity labeling and ultracentrifugation experiments. Gel filtration data also suggested the presence of a third binding site of 5.4 kDa. The 170-kDa site had a binding affinity of Kd = 12 x 10(-6) and bound both LH and hCG. The small molecular weight site had an affinity of Kd = 9.4 x 10(-8) and was CG specific. Collectively, these data demonstrate the presence of multiple hormone binding sites in X. maltophilia that differ in molecular size, binding affinity, and ligand specificity.     

Skin graft survival in genetically identical cloned pigs

Nuclear transfer technology allows for the reprogramming of somatic cells, and the production of embryonic stem cells and animals that are genetically identical in terms of nuclear DNA to the parental somatic cell. It is assumed that these products of nuclear transfer technology will be immunologically compatible to each other in spite of the fact that there are data that show differences in the expression patterns and phenotypes between animals produced by nuclear transfer. We have produced a series of cloned pigs from embryonic fibroblasts. Microsatellite analysis was used to confirm that the clones were genetically identical. Skin transplants were performed to assess immunological reactivity. Skin transplants between genetically identical cloned pigs were accepted, whereas third party grafts were rejected. Histological analysis of the grafts showed edema and mononuclear cell infiltrates in the recipient's skin in rejected grafts and not in grafts that were accepted. Our data supports the notion that genetically identical cloned pigs are immunologically compatible.     

Rolling adhesion kinematics of yeast engineered to express selectins

Selectins are cell adhesion molecules that mediate capture of leukocytes on vascular endothelium as an essential component of the inflammatory response. Here we describe a method for yeast surface display of selectins, together with a functional assay that measures rolling adhesion of selectin-expressing yeast on a ligand-coated surface. E-selectin-expressing yeast roll specifically on surfaces bearing sialyl-Lewis-x ligands. Observation of yeast rolling dynamics at various stages of their life cycle indicates that the kinematics of yeast motion depends on the ratio of the bud radius to the parent radius (B/P). Large-budded yeast "walk" across the surface, alternately pivoting about bud and parent. Small-budded yeast "wobble" across the surface, with bud pivoting about parent. Tracking the bud location of budding yeast allows measurement of the angular velocity of the yeast particle. Comparison of translational and angular velocities of budding yeast demonstrates that selectin-expressing cells are rolling rather than slipping across ligand-coated surfaces.     

The MUR3 gene of Arabidopsis encodes a xyloglucan galactosyltransferase that is evolutionarily related to animal exostosins

Xyloglucans are the principal glycans that interlace cellulose microfibrils in most flowering plants. The mur3 mutant of Arabidopsis contains a severely altered structure of this polysaccharide because of the absence of a conserved alpha-L-fucosyl-(1-->2)-beta-D-galactosyl side chain and excessive galactosylation at an alternative xylose residue. Despite this severe structural alteration, mur3 plants were phenotypically normal and exhibited tensile strength in their inflorescence stems comparable to that of wild-type plants. The MUR3 gene was cloned positionally and shown to encode a xyloglucan galactosyltransferase that acts specifically on the third xylose residue within the XXXG core structure of xyloglucan. MUR3 belongs to a large family of type-II membrane proteins that is evolutionarily conserved among higher plants. The enzyme shows sequence similarities to the glucuronosyltransferase domain of exostosins, a class of animal glycosyltransferases that catalyze the synthesis of heparan sulfate, a glycosaminoglycan with numerous roles in cell differentiation and development. This finding suggests that components of the plant cell wall and of the animal extracellular matrix are synthesized by evolutionarily related enzymes even though the structures of the corresponding polysaccharides are entirely different from each other.     

Phylogenetic analysis of Blattabacterium,endosymbiotic bacteria from the wood roach,Cryptocercus (Blattodea: Cryptocercidae), including a description of three new species

Members of the cockroach genus Cryptocercus are wood-feeding, subsocial insects that live in temperate forests of the Nearctic and Palaearctic. At present, nine species are recognized: Cryptocercus relictus and Cryptocercus kyebangensis in eastern Asia and Russia, Cryptocercus primarius and Cryptocercus matilei in southwestern China, Cryptocercus clevelandi in the western USA, and Cryptocercus darwini, Cryptocercus garciai, Cryptocercus punctulatus, and Cryptocercus wrighti in the eastern USA. Like all extant cockroaches, Cryptocercus harbor endosymbiotic bacteria, Blattabacterium, in their fat bodies. The endosymbionts in all cockroaches have been considered a single species, Blattabacterium cuenoti, since their discovery about a century ago. However, a recent analysis of DNA sequences from representatives of four cockroach families has indicated that there is considerable DNA sequence divergence among B. cuenoti from different host species. As a part of our studies on the evolution of Cryptocercus, we examined DNA sequence divergence among B. cuenoti from six of the nine known Cryptocercus species. Specifically, we sequenced approximately 2,400 bp of the 16S rRNA and 23S rRNA genes of B. cuenoti from six species of Cryptocercus. We found that B. cuenoti in Cryptocercus has differentiated into multiple monophyletic lineages distinguishable by DNA sequence of rRNA genes and host association. Our sequence divergence estimates were consistent with those reported for other, congeneric bacterial species. We propose the recognition of three new species of Blattabacterium within Cryptocercus species as follows: Blattabacterium relictus sp. nov. in C. relictus, Blattabacterium clevelandi sp. nov. in C. clevelandi, and Blattabacterium punctulatus sp. nov. in C. darwini, C. garciai, C. punctulatus, and C. wrighti.     

The effect of liquid carbohydrate ingestion on repeated maximal effort exercise in competitive cyclists

We investigated the effects of carbohydrate ingestion during recovery from high-intensity exercise on subsequent high-intensity exercise in trained cyclists. Aerobic power was determined, and the competitive cyclists (N = 7) were familiarized with the 100-kJ test protocol (100 KJ-TEST). The subjects performed a first 100 KJ-TEST (RIDE-1), ingested 0.7 g.(kg body mass)(-1) of Gatorlode (CHO) or placebo (PLC), rested for 60 minutes, and then performed a second 100 KJ-TEST (RIDE-2). Blood samples taken before (PRE-1) and after (POST-1) RIDE-1 and before (PRE-2) and after (POST-2) RIDE-2 were analyzed for plasma glucose ([glucose]), lactate, and nonesterified fatty acids ([NEFA]). No significant differences (p > 0.05) were observed between treatments in time to complete RIDE-1 (CHO = 270.3 +/- 29.0 seconds; PLC = 269.9 +/- 33.0 seconds) and RIDE-2 (CHO = 271.7 +/- 26.6 seconds; PLC = 275.3 +/- 30.6 seconds). Plasma [glucose] significantly decreased during the 60-minute recovery for PLC. There was an interaction effect for [NEFA] during recovery, with [NEFA] increasing for PLC and decreasing for CHO. Carbohydrate ingestion after maximal exercise does not appear to influence subsequent short-duration maximal effort exercise in competitive cyclists but does alter plasma [glucose] and [NEFA] relative to a PLC condition.     

Lipid Raft targeting of the TC10 amino terminal domain is responsible for disruption of adipocyte cortical actin

Overexpression of the Rho family member TC10alpha, disrupts adipocyte cortical actin structure and inhibits insulin-stimulated GLUT4 translocation when targeted to lipid raft microdomains. This appears to be independent of effecter domain function because overexpression of the wild-type (TC10/WT), constitutively GTP-bound (TC10/Q75L), and constitutively GDP bound (TC10/T31N) all inhibit adipocyte cortical actin structure and GLUT4 translocation. To examine the structural determinants responsible for these effects, we generated a series of chimera proteins between TC10 with that of H-Ras and K-Ras. Chimera containing the 79 (TC10-79/H-Ras), 41 (TC10-41/H-Ras), or 16 (TC10-16/H-Ras) amino acids of the TC10 amino terminal extension fused to H-Ras disrupted cortical actin and inhibited insulin-stimulated GLUT4 translocation. In contrast, the same amino terminal TC10 extensions fused to K-Ras had no significant effect on either GLUT4 translocation or cortical actin structure. Similarly, expression of TC10beta was without effect, whereas fusion of the amino terminal 8 amino acid of TC10alpha onto TC10beta resulted in an inhibition of insulin-stimulated GLUT4 translocation. Within the amino terminal extension point mutation analysis demonstrated that both a GAG and GPG sequences when lipid raft targeted was essential for these effects. Furthermore, expression of the amino terminal TC10 deletions DeltaNT-TC10/WT or DeltaNT-TC10/T31N had no detectable effect on cortical actin organization and did not perturb insulin-stimulated GLUT4 translocation. Surprisingly, however, expression of DeltaNT-TC10/Q75L remained fully capable of inhibiting insulin-stimulated GLUT4 translocation without affecting cortical actin. These data demonstrate that inhibitory effect of TC10 overexpression on adipocyte cortical actin organization is due to the specific lipid raft targeting of the unusual TC10 amino terminal extension.