A Novel Non-Lens βγ?Crystallin and Trefoil Factor Complex from Amphibian Skin and Its Functional Implications

Background

In vertebrates, non-lens βγ-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive.

Principal Findings

A naturally existing 72-kDa complex of non-lens βγ-crystallin (α-subunit) and trefoil factor (β-subunit), named βγ-CAT, was identified from frog Bombina maxima skin secretions. Its α-subunit and β-subunit (containing three trefoil factor domains), with a non-covalently linked form of αβ₂, show significant sequence homology to ep37 proteins, a group of non-lens βγ-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. βγ-CAT showed potent hemolytic activity on mammalian erythrocytes. The specific antiserum against each subunit was able to neutralize its hemolytic activity, indicating that the two subunits are functionally associated. βγ-CAT formed membrane pores with a functional diameter about 2.0 nm, leading to K⁺ efflux and colloid-osmotic hemolysis. High molecular weight SDS-stable oligomers (>240-kDa) were detected by antibodies against the α-subunit with Western blotting. Furthermore, βγ-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of βγ-CAT (25–50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC₅₀ 10 nM) and apoptosis. βγ-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. Bafilomycin A1 (a specific inhibitor of the vacuolar-type ATPase) and nocodazole (an agent of microtuble depolymerizing), while inhibited βγ-CAT induced vacuole formation, significantly inhibited βγ-CAT induced cell detachment, suggesting that βγ-CAT endocytosis is important for its activities.

Conclusions/Significance

These findings illustrate novel cellular functions of non-lens βγ-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals.     

A novel mechanism by which thiazolidinediones facilitate the proteasomal degradation of cyclin D1 in cancer cells

This study identifies a novel mechanism by which thiazolidinediones mediate cyclin D1 repression in prostate cancer cells. Based on the finding that the thiazolidinedione family of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists mediated PPARgamma-independent cyclin D1 degradation, we developed a novel PPARgamma-inactive troglitazone derivative, STG28, with high potency in cyclin D1 ablation. STG28-mediated cyclin D1 degradation was preceded by Thr-286 phosphorylation and nuclear export, which however, were independent of glycogen synthase kinase 3beta. Mutational analysis further confirmed the pivotal role of Thr-286 phosphorylation in STG28-induced nuclear export and proteolysis. Of several kinases examined, inhibition of IkappaB kinase alpha blocked STG28-mediated cytoplasmic sequestration and degradation of cyclin D1. Pulldown of ectopically expressed Cul1, the scaffold protein of the Skp-Cullin-F-box E3 ligase, in STG28-treated cells revealed an increased association of cyclin D1 with beta-TrCP, whereas no specific binding was noted with other F-box proteins examined, including Skp2, Fbw7, Fbx4, and Fbxw8. This finding represents the first evidence that cyclin D1 is targeted by beta-TrCP. Moreover, beta-TrCP expression was up-regulated in response to STG28, and ectopic expression and small interfering RNA-mediated knock-down of beta-TrCP enhanced and protected against STG28-facilitated cyclin D1 degradation, respectively. Because cyclin D1 lacks the DSG destruction motif, mutational and modeling analyses indicate that cyclin D1 was targeted by beta-TrCP through an unconventional recognition site, (279)EEVDLACpT(286), reminiscent to that of Wee1. Moreover, we obtained evidence that this beta-TrCP-dependent degradation takes part in controlling cyclin D1 turnover when cancer cells undergo glucose starvation, which endows physiological relevance to this novel mechanism.     

Detection of tripping gait patterns in the elderly using autoregressive features and support vector machines

Elderly tripping falls cost billions annually in medical funds and result in high mortality rates often perpetrated by pulmonary embolism (internal bleeding) and infected fractures that do not heal well. In this paper, we propose an intelligent gait detection system (AR-SVM) for screening elderly individuals at risk of suffering tripping falls. The motivation of this system is to provide early detection of elderly gait reminiscent of tripping characteristics so that preventive measures could be administered. Our system is composed of two stages, a predictor model estimated by an autoregressive (AR) process and a support vector machine (SVM) classifier. The system input is a digital signal constructed from consecutive measurements of minimum toe clearance (MTC) representative of steady-state walking. The AR-SVM system was tested on 23 individuals (13 healthy and 10 having suffered at least one tripping fall in the past year) who each completed a minimum of 10 min of walking on a treadmill at a self-selected pace. In the first stage, a fourth order AR model required at least 64 MTC values to correctly detect all fallers and non-fallers. Detection was further improved to less than 1 min of walking when the model coefficients were used as input features to the SVM classifier. The system achieved a detection accuracy of 95.65% with the leave one out method using only 16 MTC samples, but was reduced to 69.57% when eight MTC samples were used. These results demonstrate a fast and efficient system requiring a small number of strides and only MTC measurements for accurate detection of tripping gait characteristics.     

Protein markers in the microcyst of the posteroventral cochlear nucleus of the gerbil

Microcysts are most evident in the posteroventral and anteroventral cochlear nuclei (PVCN and AVCN) of the Mongolian gerbil. The origin and contents of the microcyst are not elucidated at present. The present study investigated the possible inclusions in the microcyst by employing immunocytochemical labeling to localize the existence of various protein markers. Thirty and 100 microm thick sections were used to substitute and reconstruct between 6 and 20 paraffin serial sections, respectively. In 30-microm-thick slice sections, immunoreactivity of glial fibrillary acidic protein (GFAP-IR), mitochondria inner membrane (MCA-In-IR), S-100 (S-100-IR), serotonin (5-HT-IR), myelin proteolipid protein (PLP-IR) and substance P (SP-IR) abutted on the perimeter of the microcyst. The immunolabeled SP-positive cells were adjacent to the evagination of the microcyst. In 100-microm-thick slice sections, immunoreactivity of nitric oxide synthase (NOS-IR) and somatostatin (SOM-IR) mainly precipitated as flocculent structures in the small to medium-sized microcysts. 5-HT-IR also precipitated as an elongated flocculent stalk adjacent to the large microcyst or randomly distributed in the neuropil. The findings suggest that GFAP, MCA-In, S-100, 5-HT, PLP, SP, NOS and SOM may be involved in modulating the physiological functions and maintaining micro-environmental homeostasis of the microcyst in the cochlear nucleus of the gerbil.     

Morphological observation of nuclear sub-localization of human angiogenin in HUVECs

Angiogenin, a potent angiogenic factor, was cloned and expressed by Escherichia coli and then purified with gel filtration chromatography. Approximately 90% pure angiogenin was obtained to generate a monoclonal antibody. Using western immunoblotting and ELISA, we confirmed that monoclonal antibody C46 secreted from hybridoma cells stably and specifically binds to angiogenin. The fused protein angiogenin-EGF was then expressed in HUVECs, and the subcellular localization of the recombinant protein was determined by confocal microscopy and TEM assay. Recombinant angiogenin was found to mainly concentrate in the pars granulosa of the nucleus, where the protein accumulates to form ribonucleoprotein particles.     

Apoptosis in meningoencephalitis of Angiostrongylus cantonensis-infected mice

A hallmark of eosinophilic meningoencephalitis is infiltration of leukocytes into brain parenchyma and subarachnoid space infected by Angiostrongylus cantonensis. Apoptosis, a process that eliminates useless cells and counterbalances tissue homeostasis, is important for homeostasis of the immune system. In this study, we investigated the characteristics of cell death induced in BABL/c mice infected with A. cantonensis. We observed increased expression of the apoptotic proteins, caspase-3, caspase-8, caspase-9, and cytochrome c, and decreased expression of anti-apoptotic proteins, B-cell leukemia 2 and inhibitor of apoptosis protein 1. On immunohistochemistry, apoptotic proteins were localized within the leukocytes infiltrate. A terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling assay to detect DNA fragmentation confirmed these observations. The infiltration of leukocytes present in the brain parenchyma and subarachnoid space in vivo may also express these apoptotic regulatory molecules, which demonstrates the capacity of these cells to undergo apoptosis.     

Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1

Molecular cues, such as netrin 1, guide axons by influencing growth cone motility. Rho GTPases are a family of intracellular proteins that regulate the cytoskeleton, substrate adhesion and vesicle trafficking. Activation of the RhoA subfamily of Rho GTPases is essential for chemorepellent axon guidance; however, their role during axonal chemoattraction is unclear. Here, we show that netrin 1, through its receptor DCC, inhibits RhoA in embryonic spinal commissural neurons. To determine whether netrin 1-mediated chemoattraction requires Rho function, we inhibited Rho signaling and assayed axon outgrowth and turning towards netrin 1. Additionally, we examined two important mechanisms that influence the guidance of axons to netrin 1: substrate adhesion and transport of the netrin receptor DCC to the plasma membrane. We found that inhibiting Rho signaling increased plasma membrane DCC and adhesion to substrate-bound netrin 1, and also enhanced netrin 1-mediated axon outgrowth and chemoattractive axon turning. Conversely, overexpression of RhoA or constitutively active RhoA inhibited axonal responses to netrin 1. These findings provide evidence that Rho signaling reduces axonal chemoattraction to netrin 1 by limiting the amount of plasma membrane DCC at the growth cone, and suggest that netrin 1-mediated inhibition of RhoA activates a positive-feedback mechanism that facilitates chemoattraction to netrin 1. Notably, these findings also have relevance for CNS regeneration research. Inhibiting RhoA promotes axon regeneration by disrupting inhibitory responses to myelin and the glial scar. By contrast, we demonstrate that axon chemoattraction to netrin 1 is not only maintained but enhanced, suggesting that this might facilitate directing regenerating axons to appropriate targets.     

Characterization of porous poly(D,L-lactic-co-glycolic acid) sponges fabricated by supercritical CO2 gas-foaming method as a scaffold for three-dimensional growth of Hep3B cells

This study presents the application of the porous poly(D,L-lactic-co-glycolic acid) (PLGA) sponges fabricated from an organic solvent free supercritical gas foaming technique. Two formulations of PLGA sponges with different co-polymer compositions (85:15 and 50:50) were fabricated as novel scaffolds to guide human hepatoma cell line, Hep3B cell growth in vitro. The PLGA sponges showed desirable biodegradability and exhibited uniform pore size distribution with moderate interconnectivity. It was observed in this study that cells cultured on PLGA sponges showed lower proliferation rate as compared to the control during 14 days of culture as measured by using total DNA and methylthiazol tetrazolium (MTT) assays. However, the cells cultured on the sponges tended to aggregate to form cell islets which were able to express better hepatic functions. The enzyme-linked immunosorbent assay (ELISA) results showed that the cell-sponge constructs secreted 1.5-3.0 times more albumin than the control when normalized to cellular content. In a similar fashion, its detoxification ability was also predominantly higher than that of the control as indicated by the ethoxyresorufin-O-deethylase (EROD) results. By comparing the cells growing on the two formulations of PLGA sponges, it was found that the PLGA 85:15 sponge exhibited better conductive and desirable environment for hep3B cells as justified by better cell infiltration, higher proliferation and hepatic function than the PLGA 50:50 sponge.