Expression of capsule-associated genes of Cryptococcus neoformans

Cryptococcus neoformans produces an extracellular polysaccharide capsule that is related to its virulence. The production of capsular components was reported to be accelerated when cultured on media with lower amount of glucose. In this study, relationship between capsule synthesis and expression of capsule-associated genes (CAP genes) was investigated by quantitative real-time PCR analysis. Normally encapsulated strains and a stable acapsular strain were cultured in 1% polypepton medium with 0.1% or 15% glucose. The results of assessment of the capsule size showed that the capsule of yeast cells cultured in the medium with low amount of glucose was thicker than that with high amount of glucose. The CAP gene expressions of normally encapsulated strains were higher in the medium with 0.1% glucose than in the medium with 15% glucose. Furthermore, CAP10, CAP59 and CAP60 genes were expressed very low in a stable acapsular strain, and CAP64 gene was not expressed. Results of assessment of capsule size and CAP gene expressions by quantitative real-time PCR analysis indicated that CAP gene expressions might be related to the production of capsule, and that glucose concentration in culture media might be related to the expression of CAP genes.     

Non-canonical functions of hunchback in segment patterning of the intermediate germ cricket Gryllus bimaculatus

In short and intermediate germ insects, only the anterior segments are specified during the blastoderm stage, leaving the posterior segments to be specified later, during embryogenesis, which differs from the segmentation process in Drosophila, a long germ insect. To elucidate the segmentation mechanisms of short and intermediate germ insects, we have investigated the orthologs of the Drosophila segmentation genes in a phylogenetically basal, intermediate germ insect, Gryllus bimaculatus (Gb). Here, we have focused on its hunchback ortholog (Gb'hb), because Drosophila hb functions as a gap gene during anterior segmentation, referred as a canonical function. Gb'hb is expressed in a gap pattern during the early stages of embryogenesis, and later in the posterior growth zone. By means of embryonic and parental RNA interference for Gb'hb, we found the following: (1) Gb'hb regulates Hox gene expression to specify regional identity in the anterior region, as observed in Drosophila and Oncopeltus; (2) Gb'hb controls germband morphogenesis and segmentation of the anterior region, probably through the pair-rule gene, even-skipped at least; (3) Gb'hb may act as a gap gene in a limited region between the posterior of the prothoracic segment and the anterior of the mesothoracic segment; and (4) Gb'hb is involved in the formation of at least seven abdominal segments, probably through its expression in the posterior growth zone, which is not conserved in Drosophila. These findings suggest that Gb'hb functions in a non-canonical manner in segment patterning. A comparison of our results with the results for other derived species revealed that the canonical hb function may have evolved from the non-canonical hb functions during evolution.     

High level expression and preparation of autonomous Ca2+/calmodulin-dependent protein kinase II in Escherichia coli

The chymotryptic fragment of Ca2+/calmodulin-dependent protein kinase II (30K-CaMKII) is a constitutively active enzyme that phosphorylates a variety of protein substrates in vitro. Although 30K-CaMKII is an often used and powerful tool for protein phosphorylation, the efficient production of catalytically active 30K-CaMKII in Escherichia coli has not yet been successfully realized, probably due to its toxicity in host cells. In this study, we found that a high-level expression of 30K-CaMKII as an insoluble form was attained when the N-terminal 43 amino acid residues of Xenopus CaMKI were fused to the N-terminal end of 30K-CaMKII (CX-30K-CaMKII). The inactive CX-30K-CaMKII thus expressed in E. coli was reactivated by simple denaturation/renaturation processes and purified on a Ni2+-chelating column. The renatured CX-30K-CaMKII exhibited specific activity similar to that of rat brain CaMKII, and phosphorylated various proteins such as histones, myosin light chain, myelin basic protein, and synapsin I, as in case of 30K-CaMKII or purified CaMKII. Thus, CX-30K-CaMKII, an autonomous CaMKII, can be obtained with a simple procedure using E. coli expression system.     

Involvement of hedgehog,wingless, and dpp in the initiation of proximodistal axis formation during the regeneration of insect legs,a verification of the modified boundary model

To understand the mechanism of regeneration, many experiments have been carried out with hemimetabolous insects, since their nymphs possess the ability to regenerate amputated legs. We first succeeded in observing expression patterns of hedgehog, wingless (wg), and decapentaplegic (dpp) during leg regeneration of the cricket Gryllus bimaculatus. The observed expression patterns were essentially consistent with the predictions derived from the boundary model modified by Campbell and Tomlinson (CTBM). Thus, we concluded that the formation of the proximodistal axis of a regenerating leg is triggered at a site where ventral wg-expressing cells abut dorsal dpp-expressing cells in the anteroposterior (A/P) boundary, as postulated in the CTBM.     

Correlation of expression patterns of homothorax, dachshund, and Distal-less with the proximodistal segmentation of the cricket leg bud

We describe the expression pattern of Gryllus homothorax (Gbhth) and dachshund (Gbdac), a cricket homologue of Drosophila homothorax and dachshund, together with localization of Distal-less or Extradenticle protein during leg development. We correlated their expression patterns with the morphological segmentation of the leg bud. The boundary of Gbhth/GbDll subdivision is correlated with the segment boundary of the future trochanter/femur at early stages. Gbdac expression subdivides the leg bud into the presumptive femur and more distal region. During the leg proximodistal formation, although the early expression patterns of GbDll, Gbdac, and Gbhth significantly differ from those of Drosophila imaginal disc, their expression patterns in the fully segmented Gryllus leg were similar to those in the Drosophila late third instar disc.     

An insulator element from the sea urchin Hemicentrotus pulcherrimus suppresses variation in transgene expression in cultured tobacco cells

Specialized DNA sequences known as insulators protect genes from both the positive and negative influences of nearby chromatin. Many insulators have been identified in various species; however, few function in multiple species. We have shown that an insulator from the Ars (arylsulfatase) gene of the sea urchin Hemicentrotus pulcherrimus functions in plant cells. Normally, expression of an introduced chimeric GUS gene is inactivated in approximately 30% of transformed tobacco BY2 clones. Transgenes containing the Ars insulator, however, were expressed in all transformed tobacco BY2 cells. The insulator did not affect the copy number, the chromosomal position of transgene integration or maximum expression levels. These results suggest that the insulator functions to suppress the variation normally associated with transgene expression in tobacco BY2 cells.     

Suitability of poaceous plants for nymphal growth of the pecky rice bugs Trigonotylus caelestialium and Stenotus rubrovittatus (Hemiptera: Miridae) in Niigata, Japan

Under laboratory conditions, we investigated the host suitability of 24 poaceous plants for Trigonotylus caelestialium (Kirkaldy) and Stenotus rubrovittatus (Matsumura). More than 50?% of the nymphs of both bugs reached the adult stage on Poa annua L., Alopecurus aequalis Sobol. var. amurensis (Komar.), Poa sphondylodes Trin., Hordeum murinum L., Agrostis clavata Trin. ssp. matsumurae (Hack. ex Honda), and Lolium multiflorum Lam. In addition, a similar number of S. rubrovittatus nymphs reached the adult stage on Dactylis glomerata L. and Digitaria violascens Link. However, a high percentage of T. caelestialium adults emerged on both spikelets and leaves of the host plants, whereas a lower percentage of S. rubrovittatus adults emerged on leaves than on spikelets. While the numbers of T. caelestialium adults that emerged on spikelets and leaves were similar, those reared on spikelets had a shorter developmental period and longer forewings than those reared on leaves, indicating that spikelets were more suitable for growth. In addition, more adults of both species emerged on plants with ears that arise during the spring and early summer than on plants with ears that arise during the summer and fall. Therefore, the development of these bugs is dependent on the season of ear emergence.     

A 42-year-old patient presenting with femoral head migration after hemiarthroplasty performed 22 years earlier: a case report

Introduction

Treatment of femoral neck fractures in young adults may require total hip arthroplasty or hip hemiarthroplasty using a bipolar cup. The latter can, however, result in migration of the femoral head and poor long-term results.

Case presentation

We report a case of femoral head migration after hemiarthroplasty performed for femoral neck fracture that had occurred 22 years earlier, when the patient (a Japanese man) was 20 years old. He experienced peri-prosthetic fracture of the femur, subsequent migration of the prosthesis, and a massive bone defect of the pelvic side acetabular roof. After bone union of the femoral shaft fracture, the patient was referred to our hospital for reconstruction of the acetabular roof. Intra-operatively, we placed two alloimplants of bone from around the transplanted femoral head into the weight-bearing region of the acetabular roof using an impaction bone graft method. We then implanted an acetabular roof reinforcement plate and a cemented polyethylene cup in the position of the original acetabular cup. Eighteen months post-operatively, X-rays showed union of the transplanted bone.

Conclusions

Treatment of femoral neck fractures in young adults is usually accomplished by osteosynthesis, but it may be complicated by femoral head avascular necrosis or by infection or osteomyelitis. In such cases, once an infection has subsided, either hip hemiarthroplasty using a bipolar cup or total hip arthroplasty may be required. However, if the acetabular side articular cartilage is damaged, a bipolar cup should not be used. Total hip arthroplasty should be performed to prevent migration of the implant.     

The clinical significance of 5% change in vital capacity in patients with idiopathic pulmonary fibrosis: extended analysis of the pirfenidone trial

Background

The rate of decline in forced expiratory volume in 1 second (FEV₁) is representative of the natural history of COPD. Sparse information exists regarding the associations between the magnitude of annualised loss of FEV₁ with other endpoints.

Methods

Retrospective analysis of UPLIFT^® trial (four-year, randomized, double-blind, placebo-controlled trial of tiotropium 18 μg daily in chronic obstructive pulmonary disease [COPD], n = 5993). Decline of FEV₁ was analysed with random co-efficient regression. Patients were categorised according to quartiles based on the rate of decline (RoD) in post-bronchodilator FEV_1. The St George's Respiratory Questionnaire (SGRQ) total score, exacerbations and mortality were assessed within each quartile.

Results

Mean (standard error [SE]) post-bronchodilator FEV₁ increased in the first quartile (Q1) by 37 (1) mL/year. The other quartiles showed annualised declines in FEV₁ (mL/year) as follows: Q2 = 24 (1), Q3 = 59 (1) and Q4 = 125 (2). Age, gender, respiratory medication use at baseline and SGRQ did not distinguish groups. The patient subgroup with the largest RoD had less severe lung disease at baseline and contained a higher proportion of current smokers. The percentage of patients with ≥ 1 exacerbation showed a minimal difference from the lowest to the largest RoD, but exacerbation rates increased with increasing RoD. The highest proportion of patients with ≥ 1 hospitalised exacerbation was in Q4 (Q1 = 19.5% [tiotropium], 26% [control]; Q4 = 33.8% [tiotropium] and 33.1% [control]). Time to first exacerbation and hospitalised exacerbation was shorter with increasing RoD. Rate of decline in SGRQ increased in direct proportion to each quartile. The group with the largest RoD had the highest mortality.

Conclusion

Patients can be grouped into different RoD quartiles with the observation of different clinical outcomes indicating that specific (or more aggressive) approaches to management may be needed.

Trial Registration

ClinicalTrials.gov number, NCT00144339