A femoral neck fracture model in rabbits

A technique was developed to create a reproducible femoral neck fracture in vitro using 5-month-old JW/CSK series male rabbits. Force attenuation of a newly developed damping material was also evaluated using this model. Ten pairs of the femora with smaller deviations in length and weight were harvested and cleaned of soft tissue. Either a right or left of each pair of the specimens was randomly selected and put into either the control or the experimental group, both of which contained equal numbers of the right and left femora. The specimens were attached to an L-shaped plate and embedded in a resin from the proximal diaphysis to the distal end so as to maintain a consistent position of the femora. They were mounted and fixed on a pedestal slanted in the coronal plane at 20 degrees. The impact load testing was conducted using an impact mallet dropped from a height of 3 cm. The impact load was applied onto the femoral head. To the specimens in the experimental group, attenuated impact forces were loaded through the damping material, but those in the control group were subjected to forces directly transmitted without the material. All the impact testing was performed in a temperature and humidity controlled chamber. All of the femoral specimens exposed to the direct impact forces (controlled group) sustained fracture at the neck. The fracture line passed from the base of the femoral head laterally and to the calcar area just proximal to the minor trochanter medially. The location of each fracture line was almost identical among the specimens. None of the specimens that were exposed to the impact force through the damping material (experimental group) sustained fracture macroscopically and roentgenographically.     

Characterization of nucleotide pyrophosphatase-5 as an oligomannosidic glycoprotein in rat brain

Membrane glycoproteins of neural cells play crucial roles in axon guidance, synaptogenesis, and neuronal transmission. We have here characterized membrane glycoproteins containing terminal alpha-mannose residues in rat brain membranes. Affinity purification using Galanthus nivalis agglutinin, that is highly specific for terminal alpha-mannose residues, revealed a 50-kDa protein as well as 80-kDa SHPS-1 and 45-kDa beta2 subunit of Na,K-ATPase in rat brain membranes. Combination of N-terminal peptide sequencing and mass spectrometry indicated that the 50-kDa protein was rat nucleotide pyrophosphatase-5 (NPP-5). In contrast to other NPPs, NPP-5 was a type-I transmembrane protein. Northern blot analysis showed that NPP-5 was highly expressed in brain, but also expressed in other peripheral tissues. However, we could not detect either the NPP activity or the lysophospholipase D activity in the immunoprecipitates with antibodies to NPP-5 from rat brain membranes. These data, therefore, suggest that NPP-5 is a neural oligomannosidic glycoprotein that may participate in neural cell communications.     

Spatial pattern of allozyme variation in a contact zone of Pinus Ponderosa and P. arizonica (Pinaceae)

The spatial distribution of genotypes for nine polymorphic allozyme loci was examined in a contact zone between Pinus ponderosa var. scopulorum and another tree regarded as either a separate species, Pinus arizonica, or variety, Pinus ponderosa var. arizonica, in southern Arizona. Previous work had identified a steep elevational cline for a key taxonomic trait, number of leaf-needles per fascicle, on the south slope of Mt. Lemmon. The present results indicate that the taxa are not fully interbreeding in this contact zone, because allozyme genotypes are considerably more spatially structured than expected for the dispersal characteristics of pines. The amount of spatial differentiation is also much less than that observed for needle number. It appears that this is due to the lack of differentiation for allozyme gene frequencies for the two types of trees, which is further evidenced by analysis of samples from two other populations away from the contact zone. It is likely that if the two taxa were isolated in the past, it was not for long enough nor complete enough to allow mutation-drift to create substantial differentiation between them. Another possible explanation is that introgression after recontact is so advanced that any differences have been erased throughout the Santa Catalina mountain range.     

Genetic structure of age classes in Camellia japonica (Theaceae)

Camellia japonica L. (Theaceae), an insect- and bird-pollinated, broad-leaved evergreen tree, is widely distributed in Japan and the southern Korean peninsula. The species has a relatively even age distribution within populations, which may influence the spatial genetic structure of different age classes relative to species with typical L-shaped age distributions. To determine whether the internal spatial genetic structure found in seedlings and young individuals carries over into adults, we used allozyme loci, F-statistics, spatial autocorrelation statistics (Moran's I), and coancestry measures to examine changes in genetic structure among seven age classes in a population (60-m x 100-m area) in southern Korea. In seedlings, weak but significant positive values of Moran's I-statistics and coancestry measures were found for distances less than 14 m, which is consistent with a mechanism of limited seed dispersal combined with overlapping seed shadows. This spatial structure, however, dissipates in older age classes, and in adults genetic variation has an essentially random spatial distribution. Morisita's index of dispersion of individuals in each age class showed that seedlings and juveniles are more highly clustered than are older individuals. These results suggest that self-thinning changes the spatial relationships of individuals, and thus genotypes. A multilocus estimate of FST (0.008) shows a small but statistically significant difference in allele frequencies among age classes. In summary, intrapopulation genetic structure within and among age classes of C. japonica was significant but weak. Despite presumably limited seed dispersal, weak spatial genetic structure in juveniles suggests overlapping seed shadows followed by self-thinning during recruitment. The present study also demonstrates that studies of spatial genetic structure focusing on limited numbers of generations may not be sufficient to reveal the entire picture of genetic structure in populations with overlapping generations.     

A novel protein phosphatase 2C family member (PP2Czeta) is able to associate with ubiquitin conjugating enzyme 9

In this study we have cloned a novel member of mouse protein phosphatase 2C family, PP2Czeta, which is composed of 507 amino acids and has a unique N-terminal region. The overall similarity of the amino acid sequence between PP2Czeta and PP2Calpha was 22%. On Northern blot analysis PP2Czeta was found to be expressed specifically in the testicular germ cells. PP2Czeta expressed in COS7 cells was able to associate with ubiquitin conjugating enzyme 9 (UBC9) and the association was enhanced by co-expression of small ubiquitin-related modifier-1 (SUMO-1), suggesting that PP2Czeta exhibits its specific role through its SUMO-induced recruitment to UBC9.     

Cinnamate:coenzyme A ligase from the filamentous bacterium streptomyces coelicolor A3(2)

4-Coumarate:coenzyme A ligase (4CL) plays a key role in phenylpropanoid metabolism, providing precursors for a large variety of important plant secondary metabolites, such as lignin, flavonoids, and phytoalexins. Although 4CLs have been believed to be specific to plants, a gene encoding a 4CL-like enzyme which shows more than 40% identity in amino acid sequence to plant 4CLs was found in the genome of the gram-positive, filamentous bacterium Streptomyces coelicolor A3(2). The recombinant enzyme, produced in Escherichia coli with a histidine tag at its N-terminal end, showed distinct 4CL activity. The optimum pH and temperature of the reaction were pH 8.0 and 30 degrees C, respectively. The K(m) value for 4-coumarate and k(cat) were determined as 131 +/- 4 micro M and 0.202 +/- 0.007 s(-1), respectively. The K(m) value was comparable to those of plant 4CLs. The substrate specificity of this enzyme was, however, distinctly different from those of plant 4CLs. The enzyme efficiently converted cinnamate (K(m), 190 +/- 2 micro M; k(cat), 0.475 +/- 0.012 s(-1)), which is a very poor substrate for plant 4CLs. Furthermore, the enzyme showed only low activity toward caffeate and no activity toward ferulate, both of which are generally good substrates for plant 4CLs. The enzyme was therefore named ScCCL for S. coelicolor A3(2) cinnamate CoA ligase. To determine the amino acid residues providing the unique substrate specificity of ScCCL, eight ScCCL mutant enzymes having a mutation(s) at amino acid residues that probably line up along the substrate-binding pocket were generated. Mutant A294G used caffeate as a substrate more efficiently than ScCCL, and mutant A294G/A318G used ferulate, which ScCCL could not use as a substrate, suggesting that Ala(294) and Ala(318) are involved in substrate recognition. Furthermore, the catalytic activities of A294G and A294G/A318G toward cinnamate and 4-coumarate were greatly enhanced compared with those of the wild-type enzyme.     

The roles of turn formation and cross-strand interactions in fibrillization of peptides derived from the OspA single-layer beta-sheet

We previously demonstrated that a beta-hairpin peptide, termed BH(9-10), derived from a single-layer beta-sheet of Borrelia OspA protein, formed a native-like beta-turn in trifluoroethanol (TFE) solution, and it assembled into amyloid-like fibrils at higher TFE concentrations. This peptide is highly charged, and fibrillization of such a hydrophilic peptide is quite unusual. In this study, we designed a circularly permutated peptide of BH(9-10), termed BH(10-9). When folded into their respective beta-hairpin structures found in OspA, these peptides would have identical cross-strand interactions but different turns connecting the strands. NMR study revealed that BH(10-9) had little propensity to form a turn structure both in aqueous and TFE solutions. At higher TFE concentration, BH(10-9) precipitated with a concomitant alpha-to-beta conformational conversion, in a similar manner to the BH(9-10) fibrillization. However, the BH(10-9) precipitates were nonfibrillar aggregation. The precipitation kinetics of BH(10-9) was exponential, consistent with a first-order molecular assembly reaction, while the fibrillization of BH(9-10) showed sigmoidal kinetics, indicative of a two-step reaction consisting of nucleation and molecular assembly. The correlation between native-like turn formation and fibrillization of our peptide system strongly suggests that BH(9-10) adopts a native-like beta-hairpin conformation in the fibrils. Remarkably, seeding with the preformed BH(10-9) precipitates changed the two-step BH(9-10) fibrillization to a one-step molecular assembly reaction, and disrupted the BH(9-10) fibril structure, indicating interactions between the BH(10-9) aggregates and the BH(9-10) peptide. Our results suggest that, in these peptides, cross-strand interactions are the driving force for molecular assembly, and turn formation limits modes of peptide assembly.     

Phylogenetic relationships among wild and cultivated Tartary buckwheat (Fagopyrum tataricum Gaert.) populations revealed by AFLP analyses

Phylogenetic relationships among cultivated landraces and natural populations of wild subspecies of Tartary buckwheat were investigated at the individual level by constructing a phylogenetic tree based on amplified fragment length polymorphism (AFLP) markers. Seven individuals from seven cultivated landraces and 35 individuals from 21 natural populations of wild subspecies were utilized for AFLP analyses. Three groups were recognized: (1) all cultivated landraces and wild subspecies from northern Pakistan, central and eastern Tibet, and northwestern Yunnan, (2) wild subspecies from central and southern Sichuan, (3) wild subspecies from northern Sichuan and eastern Tibet. It was concluded that cultivated Tartary buckwheat probably originated in eastern Tibet or northwestern Yunnan in China.     

Relationship between partial inhibition of glycolysis and hemolysis after induction of gametocytogenesis in synchronous cultures of Plasmodium falciparum

In the present study, the low molecular-weight fraction of the culture supernatant of anti-Plasmodium falciparum antibody-producing hybridoma cells (HybSL) was used in synchronous culture with P. falciparum FVO strain. When synchronous cultures were treated with HybSL solution on day 5, gametocytogenesis was also induced. Gametocytes were consistently found from the third day after treatment and reached a peak on the fourth day. An increase in pH and hemoglobin concentrations and decrease in lactate concentrations were observed on the first day after treatment. These phenomena suggested that HybSL solution partially inhibited glycolysis of erythrocytes parasitized with schizonts and resulted in hemolysis of infected erythrocytes. On the other hand, the production of gametocytes did not increase in cultures treated with HybSL solution on day 4 of synchronous cultures in which ring forms were plentiful. Most ring forms were not killed by HybSL solution and quickly developed to trophozoites and schizonts rather than gametocytes. Consequently, it is assumed that ring forms on day 4 of synchronous cultures have finished differentiation into the asexual stage. The conversion of asexual parasites to gametocytes may be triggered only when late-stage trophozoites or early-stage schizonts are treated with HybSL solution.     

A calcium-binding protein with four EF-hand motifs in Streptomyces ambofaciens

A gene (cabA) encoding a calcium-binding protein was cloned from Streptomyces ambofaciens. CabA was 180 amino acid residues long and contained four typical EF-hand motifs bearing high sequence similarity to the calcium-binding sites in calmodulin. Consistent with this, CabA showed distinct calcium-binding activity, comparable to bovine brain calmodulin. cabA was transcribed throughout growth, as found by S1 nuclease mapping. Southern hybridization experiments showed that a single copy of cabA was present in various Streptomyces species. A hypothetical relationship between CabA and aerial mycelium formation in this strain was examined, since S. ambofaciens showed calcium-dependent aerial mycelium formation. However, disruption of cabA or overexpression of cabA in S. ambofaciens caused no detectable phenotypic changes.