Glucocorticoids maintain the extracellular matrix of differentiated mammary tissue during explant and whole organ culture

Mouse mammary whole organ culture (WOC) and explant culture of lactating tissue were used to investigate the mechanism by which glucocorticoids maintain secretory epithelium following lobuloalveolar development. The relative number of mammary epithelial cells expressing glucocorticoid receptors did not change with the loss of secretory epithelium during involution as demonstrated with competitive binding assays and immunohistochemistry for the glucocorticoid receptor. Furthermore, glucocorticoids did not inhibit AP-1 binding activity. However, Northern analysis demonstrated that genes associated with the breakdown of the extracellular matrix were not expressed in tissues cultured with glucocorticoids, in contrast to their upregulation during involution of mammary tissue cultured with insulin alone. Tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression was lowest in tissue cultured in the presence of glucocorticoids and increased 2.3-, 3.4-, and 9-fold when tissues were involuted in the presence of insulin (Ins) alone, Ins and hydrocortisone (Hyd) with 0. 005 mg/ml, or 0.01 mg/ml collagenase IV, respectively. These data indicate that glucocorticoids maintain mammary differentiation in part by inhibiting the turnover of basement membrane.     

Kainic acid-induced neurotrophic activities in developing cortical neurons

Using primary cultured cortical neurons from embryonic rat brains, we elucidated an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainic acid (KA) receptor-mediated neuroprotective mechanism through actions of nerve growth factor (NGF) in developing neurons. Neurotoxicity of KA in early days in vitro neurons was quite low compared with the mature neurons. However, pretreatment with anti-NGF antibody or TrkA inhibitor AG-879 profoundly raised KA toxicity. Furthermore, KA stimulation resulted in an increase of TrkA expression and phosphorylation, which was blocked not only by the AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and AG-879, but also by the phospholipase C inhibitor U73122 and the intracellular calcium chelator BAPTA. A study of polyphosphoinositide turnover showed that KA-stimulated phospholipase C (PLC) activity was directly triggered by the AMPA/KA receptor activity, but not by the activity of TrkA or other excitatory amino acid receptor subtypes. Sources of KA-increased intracellular calcium levels were contributed by both extracellular calcium influx and intracellular calcium release and were partially sensitive to guanosine 5'-O-(2-thiodiphosphate). These results indicate that in developing cortical neurons, activation of AMPA/KA receptors by KA may induce expression, followed by activation of TrkA via PLC signaling and intracellular calcium elevation and hence increase reception of NGF on KA-challenged neurons. A G protein-coupled AMPA/KA receptor may be involved in these metabotropic events for neuronal protection.     

Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures.

Assembly of bacteriophage P22 procapsids requires the participation of approximately 300 molecules of scaffolding protein in addition to the 420 coat protein subunits. In the absence of the scaffolding, the P22 coat protein can assemble both wild-type-size and smaller size closed capsids. Both sizes of procapsid assembled in the absence of the scaffolding protein have been studied by electron cryomicroscopy. These structural studies show that the larger capsids have T = 7 icosahedral lattices and appear the same as wild-type procapsids. The smaller capsids possess T = 4 icosahedral symmetry. The two procapsids consist of very similar penton and hexon clusters, except for an increased curvature present in the T = 4 hexon. In particular, the pronounced skewing of the hexons is conserved in both sizes of capsid. The T = 7 procapsid has a local non-icosahedral twofold axis in the center of the hexon and thus contains four unique quasi-equivalent coat protein conformations that are the same as those in the T = 4 procapsid. Models of how the scaffolding protein may direct these four coat subunit types into a T = 7 rather than a T = 4 procapsid are presented.     

Biological Reduction of Trichloroethene Supported by Fe(0)

An anaerobic culture reductively transformed trichloroethene (TCE) in an aqueous medium containing elemental iron as the sole electron source. The TCE disappearance rate was enhanced and the product distribution was markedly altered when the culture was present. In abiotic samples containing Fe(0) but no culture, 11 µmol TCE (equivalent to an aqueous concentration of 260 µM) disappeared over a period of 39 days, with ethene and ethane as the major reduction products. Small amounts of cis-dichloroethene (cis-DCE), 1,1-DCE, and vinyl chloride (VC) also were detected. When the culture was incubated with TCE and Fe(0), the same amount of TCE was transformed in less than 2 weeks. The major products after 39 days were VC, ethene, and ethane. VC accounted for 65% of the initial TCE and appeared to be reduced further to ethene at slow rates. The significant VC production in the culture-amended samples indicates that most TCE was transformed microbially rather than chemically. The data indicate that abiotic and biological reduction of chlorinated ethenes can be coupled to enhance treatment efficiency. The results also suggest that microbial dechlorination within and downgradient from iron walls is potentially important for evaluating the long-term performance of permeable iron barriers.     

Short-term effects of dam removal on macroinvertebrates in a Taiwan stream

Dam removal is an approach for restoring rivers. However, there are increasing concerns about the impact of removal on downstream biota. We examined the short-term responses of benthic macroinvertebrates and their avian predator (Brown Dipper, Cinclus pallasii Temminck) in reaches downstream of a check dam after it was removed from a mountain stream in central Taiwan. The density and taxonomic richness of downstream macroinvertebrates decreased immediately after dam removal. The decreases were associated with scouring or burial by sediments from the upstream impoundment. Ten weeks post-removal, downstream macroinvertebrate densities, although marginally recovering, remained lower than both pre-removal and upstream densities. Substantial changes in community structure were not significantly associated with an increase in the proportion of taxa with short life spans. However, this small-scale disturbance had no strong effect on the abundance of their very mobile, avian predator. This study and other studies of dam removal have found that downstream sedimentation following dam removal can reduce macroinvertebrate densities and that they may recover over time. Thus, timescale must be considered when interpreting the consequences of dam removal, especially when the long-term goal is stream restoration.     

Targeting tumour necrosis factor alleviates signs and symptoms of inflammatory osteoarthritis of the knee

Introduction

Inflammation associated with synovial expression of TNFα is a recognised feature of osteoarthritis (OA), although no studies have yet reported beneficial effects of anti-TNFα therapy on clinical manifestations of inflammation in OA.

Methods

We conducted an open-label evaluation of adalimumab over 12 weeks in 20 patients with OA of the knee and evidence of effusion clinically. Inclusion criteria included daily knee pain for the month preceding study enrolment and a summed pain score of 125 to 400 mm visual analogue scale on the Western Ontario and McMaster University Osteoarthritis Index (WOMAC) pain subscale. The primary outcome was the Osteoarthritis Research Society International/Outcome Measures in Rheumatology Clinical Trials (OARSI/OMERACT) response criterion at week 12. Secondary outcomes included the WOMAC pain score 20% and 50% improvement, WOMAC stiffness and function scores, patient and physician global visual analogue scale, as well as target joint swelling.

Results

Treatment was well tolerated and completed by 17 patients with withdrawals unrelated to lack of efficacy or adverse events. By intention to treat, an OARSI/OMERACT response was recorded in 14 (70%) patients. WOMAC pain 20% and 50% responses were recorded in 14 (70%) patients and eight (40%) patients, respectively. Significant improvement was observed in mean WOMAC pain, stiffness, function, physician and patient global, as well as target joint swelling at 12 weeks (P < 0.0001 for all). After treatment discontinuation, 16 patients were available for assessment at 22 weeks and OARSI/OMERACT response compared with baseline was still evident in 10 (50%) patients.

Conclusion

Targeting TNFα may be of therapeutic benefit in OA and requires further evaluation in controlled trials.

Trial registration

ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT00686439","term_id":"NCT00686439"}}NCT00686439.     

Caveolin‐1 down‐regulation is required for Wnt5a‐Frizzled 2 signalling in Ha‐RasV12‐induced cell transformation

Caveolin‐1 (Cav1) is down‐regulated during MK4 (MDCK cells harbouring inducible Ha‐Ras^V12 gene) transformation by Ha‐Ras^V12. Cav1 overexpression abrogates the Ha‐Ras^V12‐driven transformation of MK4 cells; however, the targeted down‐regulation of Cav1 is not sufficient to mimic this transformation. Cav1‐silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction‐related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I‐CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha‐Ras^V12‐inducing MK4 cells increased exosome‐like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I‐CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I‐CM (MK4+I‐EXs). Wnt5a, a downstream product of Ha‐Ras^V12, was markedly secreted by MK4+I‐CM and MK4+I‐EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha‐Ras^V12‐ and MK4+I‐CM‐induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down‐regulation, either by Ha‐Ras^V12 or targeted shRNA, increased frizzled‐2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I‐EXs in MDCK cells. These data suggest that Cav1‐dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha‐Ras^V12‐Wnt5a‐Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha‐Ras^V12‐driven cell transformation.     

Mitochondrial DNA phylogeography of Semisulcospira libertina (Gastropoda: Cerithioidea: Pleuroceridae): implications the history of landform changes in Taiwan

The mitochondrial DNA cytochrome c oxidase subunit I sequences from 95 specimens of Semisulcospira libertina in Taiwan were identified as two major phylogroups, exhibiting a southern and northern distribution, north of Formosa Bank and south of Miaoli Plateau. The genetic distance between these two phylogroups was 12.20 %, and the distances within-phylogroups were 4.97 and 5.56 %. According to a molecular clock of 1.56 % per lineage per million years, the divergence time between these two major phylogroups was estimated at 4.94 million years ago (mya), with the two phylogroups forming at 3.64 and 3.75 mya, respectively. Moreover, the geological events have suggested that Taiwan Island emerged above sea level at 4–5 mya, and became its present shape at 2 mya. These results suggested that these two phylogroups might originate from two independent ancestral populations or divergent before colonizing Taiwan. Within South phylogroup, the initial colonization was hypothesized to be in Kaoping River (WT), followed by its northward. The high divergence between south- and north of WT River was influenced by the formation of the Kaoping foreland basins. Within North phylogroup, the colonization was from central sub-region through paleo-Miaoli Plateau to northern and northeastern sub-regions. This study showed that the landform changes might have shaped the genetic structure of S. libertina in concert. Apparently, two cryptic species or five different genetic stocks of S. libertina could be identified; these results are useful for the evaluation and conservation of S. libertina in Taiwan.