GFP chimeric models exhibited a biphasic pattern of mesenchymal cell invasion in tendon healing

The healing of an injured musculoskeletal system requires an influx of mesenchymal cells that can differentiate into osteoblasts, fibroblasts, chondroblasts, and skeletal myoblasts. However, whether these mesenchymal cells arise from the circulation (bone marrow) or the injured tissues themselves has been controversial. To reveal the spatiotemporal characteristics of the reparative mesenchymal cells, we investigated the healing process after patellar tendon injury using two types of green fluorescent protein (GFP) chimeric rats; one expressing GFP in the circulating cells, and the other expressing it in the patellar tendon. We analyzed the behavior of GFP-positive cells after experimental tendon injury in both chimeric rats to clarify the origin of reparative cells. At 24 h after the injury, the wound contained circulation-derived cells but not tendon-derived cells. Tendon-derived cells first appeared in the wounded area at 3 days after the injury, and had significantly increased in number with time and had maintained a high level of proliferative activity until 7 days after the injury, whereas the circulation-derived cells had decreased in number and had been replaced by the tendon-derived cells. These findings suggest that circulation-derived and tendon-derived cells contribute to the healing of tendons in different periods as part of a biphasic process.     

Ectodomain shedding of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan, by TIMP-2- and TIMP-3-sensitive proteolysis

Neuroglycan C (NGC) is a transmembrane-type of chondroitin sulfate proteoglycan with an epidermal growth factor (EGF)-like module that is exclusively expressed in the CNS. Because ectodomain shedding is a common processing step for many transmembrane proteins, we examined whether NGC was subjected to proteolytic cleavage. Western blotting demonstrated the occurrence of a soluble form of NGC with a 75 kDa core glycoprotein in the soluble fraction of the young rat cerebrum. In contrast, full-length NGC with a 120 kDa core glycoprotein and its cytoplasmic fragment with a molecular size of 35 kDa could be detected in the membrane fraction. The soluble form of NGC was also detectable in culture media of fetal rat neurons, and the full-length form existed in cell layers. The amount of the soluble form in culture media was decreased by adding a physiological protease inhibitor such as a tissue inhibitor of metalloproteinase (TIMP)-2 or TIMP-3, but not by adding TIMP-1. Both EGF-like and neurite outgrowth-promoting activity of the NGC ectodomain may be regulated by this proteolytic processing.     

Auxin is required for the assembly of A-type cyclin-dependent kinase complexes in tobacco cell suspension culture

Although activation of A-type cyclin-dependent kinase (CDKA) is required for plant cell division, little is known about how CDKA is activated before commitment to cell division. Here, we show that auxin is required for the formation of active CDKA-associated complexes, promoting assembly of the complex in tobacco suspension culture Bright Yellow-2 (BY-2) cells. Protein gel blot analysis revealed that CDKA levels increased greatly after stationary-phase BY-2 cells were subcultured into fresh medium to re-enter the cell cycle. However, these increasing levels subsided when cells were subcultured into auxin-deprived medium, and a subtle increase was observed after subculturing into sucrose-deprived medium. Additionally, p13(suc1)-associated kinase activity did not increase significantly after subculturing into either auxin- or sucrose-deprived medium, but increased strongly after subculturing into medium containing both auxin and sucrose. Using gel filtration, we found that p13(suc1)-associated kinase activity against tobacco retinoblastoma-related protein was maximal in fractions corresponding to the molecular mass of the cyclin/CDKA complex. Interestingly, this peak distribution of high molecular-mass fractions of CDKA disappeared after cells were subcultured into auxin-deprived medium. These findings suggest an important role for auxin in the assembly of active CDKA-associated complexes.     

Immunological risks of adult T-cell leukemia at primary HTLV-I infection

A small percentage of human T-cell leukemia virus type-I (HTLV-I)-infected individuals develop adult T-cell leukemia (ATL). In animal experiments, inoculation of HTLV-I via the oral route, which is the main route of mother-to-child viral transmission in humans as a result of breastfeeding, induced host HTLV-I-specific T-cell unresponsiveness and resulted in increased viral load. This strongly suggested that the known epidemiological risk factors for ATL (i.e. vertical HTLV-I infection and elevated viral load) are linked by an insufficient HTLV-I-specific T-cell response. Recent findings on the anti-tumor effects of Tax-targeted vaccination in rats and the reactivation of Tax-specific T cells in ATL patients as a result of hematopoietic stem cell transplantation imply promising immunological approaches for the prophylaxis and therapy of ATL.     

The First Reported Case of Canine Subcutaneous Cryptococcus flavescens Infection

This report describes the first documented case of subcutaneous infection due to Cryptococcus flavescens in a dog. The chief symptoms of the patient dog were abscessed lesions on the dorsal muzzle, right eyelid, and lower jaw. Biopsy specimens from the lesions on the dorsal muzzle and lower jaw showed pyogranulomatous inflammation with numerous yeast cells. The patient dog was diagnosed with a subcutaneous fungal infection and orally received 5 mg/kg itraconazole once a day for 2 months, the abscesses disappeared. After 1 month at the end of treatment, the skin lesions did not redevelop. Isolates from the biopsy specimens were identified as C. flavescens by molecular analysis as well as morphologic and biochemical examination, indicating that C. flavescens is a potential canine pathogen.     

Functional processing of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N): evidence for a critical role of proteolytic processing in the regulation of its catalytic activity, subcellular localization and substrate targeting in vivo

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear homolog CaMKP-N are Ser/Thr protein phosphatases that belong to the PPM family. These phosphatases are highly specific for multifunctional CaM kinases and negatively regulate their activities. CaMKP-N is only expressed in the brain and specifically localized in the nucleus. In this study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing in both the zebrafish brain and Neuro2a cells. In Neuro2a cells, the proteolytic processing was effectively inhibited by the proteasome inhibitors MG-132, Epoxomicin, and Lactacystin, suggesting that the ubiquitin-proteasome pathway was involved in this processing. Using MG-132, we found that the proteolytic processing changed the subcellular localization of zCaMKP-N from the nucleus to the cytosol. Accompanying this change, the cellular targets of zCaMKP-N in Neuro2a cells were significantly altered. Furthermore, we obtained evidence that the zCaMKP-N activity was markedly activated when the C-terminal domain was removed by the processing. Thus, the proteolytic processing of zCaMKP-N at the C-terminal region regulates its catalytic activity, subcellular localization and substrate targeting in vivo.     

Effects of fungal culture filtrates of Verticillium lecanii (Lecanicillium spp.) hybrid strains on Heterodera glycines eggs and juveniles

Many nematode-antagonistic fungi produce secondary metabolites and enzymes that demonstrate toxicity against plant-parasitic nematodes. The objective of this study was to evaluate the effects of fungal culture filtrates of Verticillium lecanii hybrid strains on mature eggs, embryonated eggs (eggs fertilized but without development of juveniles), and second-stage juveniles (J2) of Heterodera glycines and to compare these effects with those of their parental strains. The fungal culture filtrates of certain hybrid strains inhibited egg hatch of mature eggs. Furthermore, the fungal culture filtrates of two hybrid strains, AaF23 and AaF42, exhibited high toxicity against embryonated eggs of H. glycines. However, most of the fungal culture filtrates of V. lecanii did not inactivate J2. These results suggested that enzymes or other active compounds produced by the fungal culture filtrates of V. lecanii exhibit activity against specific stages in the H. glycines life cycle. In addition, based on a visual assessment of the morphological changes in eggs caused by filtrates of each strain, there were differences between the hybrid strains and their respective parental strains with regard to the active substances produced by V. lecanii against the embryonated eggs. As a result of promoting recombination of whole genomes via protoplast fusion, several hybrid strains may have enhanced production of active substances that are different from those produced by their parental strains. It was concluded that natural substances produced by V. lecanii are one of the important factors involved in the suppression of H. glycines damage.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> RETINOBLASTOMA-RELATED PROTEIN 1 is involved in G1 phase cell cycle arrest caused by sucrose starvation

Although sucrose availability is crucial for commitment to plant cell division during G1 phase by controlling the expression of D-type cyclins, it has remained unclear how these factors mediate entry into the cell cycle. Here we show that Arabidopsis RETINOBLASTOMA-RELATED PROTEIN 1 (AtRBR1) is involved in G1-phase cell cycle arrest caused by sucrose starvation. We generated estrogen-inducible AtRBR1 RNA interference (RNAi) Arabidopsis suspension MM2d cells, and found that downregulation of AtRBR1 leads to a higher frequency of arrest in G2 phase, instead of G1-phase arrest in the uninduced control, after sucrose starvation. Synchronization experiments confirmed that downregulation of AtRBR1 leads to a prolonged G2 phase and delayed activation of G2/M marker genes. Downregulation of AtRBR1 also stimulated the activation of E2F-regulated genes when these genes were repressed in the uninduced cells under the limited sucrose conditions. We conclude that AtRBR1 is a key effector for the ability of sucrose to modulate progression from G1 phase.