WWOX dysfunction induces sequential aggregation of TRAPPC6AΔ, TIAF1, tau and amyloid ß, and causes apoptosis.

Aggregated vesicle-trafficking protein isoform TRAPPC6AΔ (TPC6AΔ) has a critical role in causing caspase activation, tau aggregation and Aß generation in the brains of nondemented middle-aged humans, patients with Alzheimer's disease (AD) and 3-week-old Wwox gene knockout mice. WWOX blocks neurodegeneration via interactions with tau and tau-phosphorylating enzymes. WWOX deficiency leads to epilepsy, mental retardation and early death. Here, we demonstrated that TGF-ß1 induces shuttling of endogenous wild-type TPC6A and TPC6AΔ in between nucleoli and mitochondria (~40-60 min per round trip), and WWOX reduces the shuttling time by 50%. TGF-ß1 initially maximizes the binding of TPC6AΔ to the C-terminal tail of WWOX, followed by dissociation. TPC6AΔ then undergoes aggregation, together with TIAF1 (TGF-ß1-induced antiapoptotic factor), in the mitochondria to induce apoptosis. An additional rescue scenario is that TGF-ß1 induces Tyr33 phosphorylation and unfolding of WWOX and its the N-terminal WW domain slowly binds TPC6AΔ to block aggregation and apoptosis. Similarly, loss of WWOX induces TPC6AΔ polymerization first, then aggregation of TIAF1, amyloid ß and tau, and subsequent cell death, suggesting that a cascade of protein aggregation leads to neurodegeneration.

WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma.

Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12-Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.

TIAF1 self-aggregation in peritumor capsule formation, spontaneous activation of SMAD-responsive promoter in p53-deficient environment, and cell death.

Self-aggregation of transforming growth factor ß (TGF-ß)1-induced antiapoptotic factor (TIAF1) is known in the nondemented human hippocampus, and the aggregating process may lead to generation of amyloid ß (Aß) for causing neurodegeneration. Here, we determined that overexpressed TIAF1 exhibits as aggregates together with Smad4 and Aß in the cancer stroma and peritumor capsules of solid tumors. Also, TIAF1/Aß aggregates are shown on the interface between brain neural cells and the metastatic cancer cell mass. TIAF1 is upregulated in developing tumors, but may disappear in established metastatic cancer cells. Growing neuroblastoma cells on the extracellular matrices from other cancer cell types induced production of aggregated TIAF1 and Aß. In vitro induction of TIAF1 self-association upregulated the expression of tumor suppressors Smad4 and WW domain-containing oxidoreductase (WOX1 or WWOX), and WOX1 in turn increased the TIAF1 expression. TIAF1/Smad4 interaction further enhanced Aß formation. TIAF1 is known to suppress SMAD-regulated promoter activation. Intriguingly, without p53, self-aggregating TIAF1 spontaneously activated the SMAD-regulated promoter. TIAF1 was essential for p53-, WOX1- and dominant-negative JNK1-induced cell death. TIAF1, p53 and WOX1 acted synergistically in suppressing anchorage-independent growth, blocking cell migration and causing apoptosis. Together, TIAF1 shows an aggregation-dependent control of tumor progression and metastasis, and regulation of cell death.

WW domain-containing oxidoreductase promotes neuronal differentiation via negative regulation of glycogen synthase kinase 3ß.

WW domain-containing oxidoreductase (WWOX), a putative tumour suppressor, is suggested to be involved in the hyperphosphorylation of Alzheimer's Tau. Tau is a microtubule-associated protein that has an important role in microtubule assembly and stability. Glycogen synthase kinase 3ß (GSK3ß) has a vital role in Tau hyperphosphorylation at its microtubule-binding domains. Hyperphosphorylated Tau has a low affinity for microtubules, thus disrupting microtubule stability. Bioinformatics analysis indicated that WWOX contains two potential GSK3ß-binding FXXXLI/VXRLE motifs. Immunofluorescence, immunoprecipitation and molecular modelling showed that WWOX interacts physically with GSK3ß. We demonstrated biochemically that WWOX can bind directly to GSK3ß through its short-chain alcohol dehydrogenase/reductase domain. Moreover, the overexpression of WWOX inhibited GSK3ß-stimulated S396 and S404 phosphorylation within the microtubule domains of Tau, indicating that WWOX is involved in regulating GSK3ß activity in cells. WWOX repressed GSK3ß activity, restored the microtubule assembly activity of Tau and promoted neurite outgrowth in SH-SY5Y cells. Conversely, RNAi-mediated knockdown of WWOX in retinoic acid (RA)-differentiated SH-SY5Y cells inhibited neurite outgrowth. These results suggest that WWOX is likely to be involved in regulating GSK3ß activity, reducing the level of phosphorylated Tau, and subsequently promoting neurite outgrowth during neuron differentiation. In summary, our data reveal a novel mechanism by which WWOX promotes neuronal differentiation in response to RA.

TGF-ß induces TIAF1 self-aggregation via type II receptor-independent signaling that leads to generation of amyloid ß plaques in Alzheimer's disease.

The role of a small transforming growth factor beta (TGF-ß)-induced TIAF1 (TGF-ß1-induced antiapoptotic factor) in the pathogenesis of Alzheimer's disease (AD) was investigated. TIAF1 physically interacts with mothers against DPP homolog 4 (Smad4), and blocks SMAD-dependent promoter activation when overexpressed. Accordingly, knockdown of TIAF1 by small interfering RNA resulted in spontaneous accumulation of Smad proteins in the nucleus and activation of the promoter governed by the SMAD complex. TGF-ß1 and environmental stress (e.g., alterations in pericellular environment) may induce TIAF1 self-aggregation in a type II TGF-ß receptor-independent manner in cells, and Smad4 interrupts the aggregation. Aggregated TIAF1 induces apoptosis in a caspase-dependent manner. By filter retardation assay, TIAF1 aggregates were found in the hippocampi of nondemented humans and AD patients. Total TIAF1-positive samples containing amyloid ß (Aß) aggregates are 17 and 48%, respectively, in the nondemented and AD groups, suggesting that TIAF1 aggregation occurs preceding formation of Aß. To test this hypothesis, in vitro analysis showed that TGF-ß-regulated TIAF1 aggregation leads to dephosphorylation of amyloid precursor protein (APP) at Thr668, followed by degradation and generation of APP intracellular domain (AICD), Aß and amyloid fibrils. Polymerized TIAF1 physically interacts with amyloid fibrils, which would favorably support plaque formation in vivo.

Light-induced retinal damage involves tyrosine 33 phosphorylation, mitochondrial and nuclear translocation of WW domain-containing oxidoreductase in vivo.

WW domain-containing oxidoreductase WOX1, also named WWOX or FOR, is a known proapoptotic protein and a candidate tumor suppressor. Stress stimuli activate WOX1 via tyrosine 33 (Tyr33) phosphorylation and translocation to the mitochondria and nuclei in vitro. Here, the potential role of WOX1 in light-induced retinal degeneration in vivo was investigated. WOX1 is expressed primarily in the inner retina at perinatal stages, whereas an enhanced expression of WOX1, along with its Tyr33 phosphorylation (p-WOX1), is shown specifically in the retinal ganglion cells in adults. Prolonged exposure of mature rats to constant, low-intensity light (500 lux) for 1-2 months resulted in substantial death of photoreceptors and the presence of activated microglia, astrocytes and Muller glial in the outer retina. However, the inner retina was not or barely affected. In the damaged inner and outer nuclear layers of rat retina, WOX1 and p-WOX1 were overly expressed. Also, WOX1 colocalized with fragments of opsin-positive cones. In rd mice with an inherited retinal deficiency, upregulation of WOX1 and p-WOX1 in degenerated retina was observed with age. By electron microscopy, a large number of immunogold particles of WOX1 and p-WOX1 were found in the damaged mitochondria and condensed nuclei of degenerating photoreceptors, indicating that WOX1 undergoes activation and translocation to these organelles. In contrast, little or no WOX1-positive particles were found in the Golgi apparatus. In conclusion, activated WOX1 is likely to exert apoptosis of neuronal cells in the outer retina during the light-induced injury and in mice with an inherited retinal defect.

Influence of maternal serum levels of vitamins C and E during the second trimester on birth weight and length.

OBJECTIVE: It has been known that maternal oxidative stress during pregnancy plays an important role in fetal growth. However, the association between antioxidant vitamin levels and birth outcomes is not conclusive. We investigated the relationship between maternal serum levels of vitamins C and E during the second trimester and birth weight and length. DESIGN: Prospective cohort study. SETTING: Outpatient-clinic of obstetrics, Ewha Womans University Hospital, South Korea. SUBJECTS AND METHODS: The study subjects were 239 healthy, pregnant women who visited an obstetric clinic for antenatal care, and their singleton live births, in Seoul, Korea, between August 2001 and March 2003. We measured the levels of vitamins C and E in maternal serum during the period 24-28 gestational weeks. Each woman was interviewed for dietary intake by trained interviewers during the second trimester. RESULTS: The serum concentration of maternal vitamin C during the second trimester was significantly associated with birth weight and length in the group of full-term deliveries. An increase of 1 microg/ml in the serum vitamin C level increased the birth weight by 27.2 g and the birth length by 0.17 cm. When we considered the levels of vitamins C and E together in the relationship with birth weight and length, we found that the heaviest birth weight and the longest birth length belonged to the group of upper vitamin C/upper vitamin E. However, dietary intake estimated by 24-h recall method was not a predictor of the levels of serum vitamins C and E. CONCLUSION: We found that maternal serum vitamin C levels during the second trimester were positively correlated with birth weight and length in full-term babies. We also found that birth weight and length were highest when the levels of both vitamins C and E were high. Our results indicate the importance of antioxidant nutrient balance for pregnant women who are exposed to various oxidants through food, drinking water, or inhaled air.

Expression of WW domain-containing oxidoreductase WOX1 in the developing murine nervous system.

WW domain-containing oxidoreductase WOX1, also known as WWOX or FOR, is a proapoptotic protein and a putative tumor suppressor. Hyaluronidases such as PH-20, Hyal-1 and Hyal-2 induce the expression of WOX1, and hyaluronidases and hyaluronan are involved in the embryonic development. In the present study, we document the expression of WOX1 in the developing murine nervous system. Immunohistochemical analysis revealed that WOX1 was differentially expressed in early dividing cells from all three germ layers from embryonic to perinatal stages. In murine fetuses, WOX1 was present prevalently in the brainstem, spinal cord and peripheral nerve bundles, but its expression decreased after birth. In parallel, the expression of WOX1, as determined by Western blotting, was significantly reduced in the brain stem and spinal cord of adult mice. Notably, high levels of WOX1 immunoreactivity was observed in the neural crest-derived structures such as cranial and spinal ganglia and cranial mesenchyme during the late fetal stage. In the adult brain, WOX1 is abundant in the epithelial cells of the choroids plexus and ependymal cells, while a low to moderate level of WOX1 is observed within white matter tracts, such as axonal profiles of the corpus callosum, striatum, optic tract, and cerebral peduncle. WOX1 is shown to mediate apoptosis synergistically with p53 in vitro. Nonetheless, the expression profiles of WOX1 were found to be similar in both p53 wild type and knockout mice, suggesting that WOX1 expression is not controlled by p53-mediated gene transcription. Taken together, in this study we have shown the expression and distribution of WOX1 in developing and adult murine nervous system. The potential role of WOX1 in the neuronal differentiation is discussed.

Acquired activated protein C resistance, high tissue factor expression, and hyper-homocysteinemia in systemic lupus erythematosus.

Activated protein C resistance (APCR), high tissue factor (TF) expression, and hyper-homocysteinemia are associated with thromboembolic diseases. Thromboembolism is a frequent complication of systemic lupus erythematosus (SLE). In this study, we evaluated the prevalence of APCR, high TF, and homocysteine with correlation of the thrombotic tendency in SLE. Ninety-four SLE patients and 28 normal controls were included. APC ratio and TF antigen were measured using commercial kits. Plasma homocysteine level was measured using HPLC. The prevalence of APCR, high TF antigen level, and hyper-homocysteinemia in our SLE patients were 21.3%, 66.0%, and 23.4%, respectively. The median plasma level of TF antigen in SLE patients was 145.23 pg/mL (range, 31.00-778.50 pg/mL), which was significantly higher than the control value of 39.83 pg/mL (range, 1.55-168.50 pg/mL). The median APC ratio in SLE patients was 2.76 (range, 1.48-13.47), which was significantly lower than the control value of 3.59 (range, 0.26-5.66). The plasma level of homocysteine was not significantly different from that of control. A significant association was observed between the presence of APCR (OR = 8.59, P < 0.0001) but not with the presence of high plasma TF antigen level (OR = 1.24, P = 0.67) and thrombotic complications in SLE patients. In conclusion, APCR and high plasma TF levels are common in SLE, but a significant association was observed only between the presence of APCR and thrombosis in SLE patients.

Hyaluronidase activation of c-Jun N-terminal kinase is necessary for protection of L929 fibrosarcoma cells from staurosporine-mediated cell death.

Hyaluronidase counteracts the growth inhibitory function of transforming growth factor beta (TGF-beta), whereas secretion of autocrine TGF-beta and hyaluronidase is necessary for progression and metastasis of various cancers. Whether hyaluronidase and TGF-beta1 induce resistance to staurosporine in L929 fibrosarcoma cells was investigated. When pretreated with TGF-beta1 for 1-2 h, L929 cells resisted staurosporine apoptosis. In contrast, without pretreatment, hyaluronidase protected L929 cells fromstaurosporine apoptosis. Hyaluronidase rapidly activated p42/44 MAPK (or ERK) in L929 cells and TGF-beta1 retarded the activation. Nonetheless, TGF-beta1 synergistically increased hyaluronidase-mediated inhibition of staurosporine apoptosis. Hyaluronidase rapidly activated c-Jun N-terminal kinase (JNK1 and JNK2) in L929 cells in 20 min. Dominant negative JNK1, JNK2, and JNK3 abolished the hyaluronidase inhibition of staurosporine apoptosis, but not the TGF-beta1 protective effect. Unlike the resistance to staurosporine, pretreatment of L929 cells with hyaluronidase is necessary to generate resistance to other anticancer drugs, including doxorubicin, daunorubicin, actinomycin D, and camptothecin, and the induced resistance was also blocked by dominant-negative JNKs. Together, hyaluronidase-mediated JNK activation is necessary to generate resistance to various anticancer drugs in L929 cells.

Hyaluronidase induction of a WW domain-containing oxidoreductase that enhances tumor necrosis factor cytotoxicity.

To determine how hyaluronidase increases certain cancer cell sensitivity to tumor necrosis factor (TNF) cytotoxicity, we report here the isolation and characterization of a hyaluronidase-induced murine WW domain-containing oxidoreductase (WOX1). WOX1 is composed of two N-terminal WW domains, a nuclear localization sequence, and a C-terminal alcohol dehydrogenase (ADH) domain. WOX1 is mainly located in the mitochondria, and the mitochondrial targeting sequence was mapped within the ADH domain. Induction of mitochondrial permeability transition by TNF, staurosporine, and atractyloside resulted in WOX1 release from mitochondria and subsequent nuclear translocation. TNF-mediated WOX1 nuclear translocation occurred shortly after that of nuclear factor-kappaB nuclear translocation, whereas both were independent events. WOX1 enhanced TNF cytotoxicity in L929 cells via its WW and ADH domains as determined using stable cell transfectants. In parallel with this observation, WOX1 also enhanced TRADD (TNF receptor-associated death domain protein)-mediated cell death in transient expression experiments. Antisense expression of WOX1 raised TNF resistance in L929 cells. Enhancement of TNF cytotoxicity by WOX1 is due, in part, to its significant down-regulation of the apoptosis inhibitors Bcl-2 and Bcl-x(L) (>85%), but up-regulation of pro-apoptotic p53 ( approximately 200%) by the ADH domain. When overexpressed, the ADH domain mediated apoptosis, probably due to modulation of expression of these proteins. The WW domains failed to modulate the expression of these proteins, but sensitized COS-7 cells to TNF killing and mediated apoptosis in various cancer cells independently of caspases. Transient cotransfection of cells with both p53 and WOX1 induced apoptosis in a synergistic manner. WOX1 colocalizes with p53 in the cytosol and binds to the proline-rich region of p53 via its WW domains. Blocking of WOX1 expression by antisense mRNA abolished p53 apoptosis. Thus, WOX1 is a mitochondrial apoptogenic protein and an essential partner of p53 in cell death.

Characterization of an apoptosis inhibitory domain at the C-termini of FE65-like protein.

TR2(L) is a 56-amino-acid polypeptide that has been shown to block TNF cytotoxicity. FE65-like (FE65L) proteins possess this conserved TR2(L) sequence at their C-termini, whereas variations in the sequences are found in the FE65 proteins. To further analyze the antiapoptotic function of TR2(L), here we utilized an isolated murine partial FE65L cDNA that encodes an N-terminal phosphotyrosine-binding domain (PTB) and the conserved C-terminal TR2(L) sequence. When L929 cells were stably transfected with the FE65L cDNA or its 3' end TR2(L) DNA sequence, these cells became resistant to TNF killing. Replacement of the N-terminal PTB domain with GFP failed to abolish the FE65L-mediated TNF resistance. Ablation of the C-terminal TR2(L) sequence through frame-shift mutation resulted in a complete loss of the FE65L function against TNF. Various protein kinase inhibitors, including lavendustin A, tyrphostin, H7, and staurosporine, which may affect the PTB domain function, could not abolish the FE65L-mediated TNF resistance. A prolonged exposure of L929 cells to these inhibitors for 24 h resulted in cell death, whereas FE65L significantly blocked the cell death. Polyclonal antibodies were generated against a synthetic peptide and shown to interact with a 38-kDa FE65L in L929 cells. Hyaluronidase downregulates the expression of FE65L gene and protein in L929 cells, and this correlates with its enhancement of TNF killing of these cells. Together, our data indicate that the TR2(L) amino acid sequence is an apoptosis-inhibitory domain commonly present in the FE65 and FE65-like family proteins.

Suppression of IkappaBalpha expression is necessary for c-Jun N-terminal kinase-mediated enhancement of Fas cytotoxicity.

The role of c-Jun N-terminal kinase (JNK) in the regulation of Fas-mediated cell death was investigated. Murine L929 fibroblasts were pretreated with anisomycin for 1 h to activate JNK, followed by exposure to anti-Fas antibodies/actinomycin D (ActD) for 16-24 h. Compared to untreated controls, the induction of JNK activation failed to raise cellular sensitivity to anti-Fas/ActD killing. Notably, a significant increase in anti-Fas/ActD killing as induced by JNK preactivation was observed in L929 cells which were engineered to suppress IkappaBalpha protein expression by antisense mRNA. Restoration of the IkappaBalpha protein level in these cells by ectopic expression of a cDNA construct abolished the JNK-increased anti-Fas/ActD killing. Despite the suppression of IkappaBalpha, no constitutive p65 (RelA) NF-kappaB nuclear translocation was observed in the IkappaBalpha-antisense cells. Also, inhibition of NF-kappaB by curcumin failed to inhibit the JNK-increased Fas cytotoxicity, suggesting that NF-kappaB is not involved in the observed effect. Most interestingly, culturing of L929 cells on extracellular protein matrices resulted in partial suppression of IkappaBalpha expression and constitutive JNK and p42/44 MAPK activation. Upon stimulation with anisomycin, these matrix protein-stimulated cells further exhibited reduced IkappaBalpha expression and p42/44 MAPK activation, as well as became sensitized to JNK-increased anti-Fas/ActD killing. Again, ectopic expression of IkappaBalpha in these cells abolished the enhanced anti-Fas/ActD killing effect. Together, these results indicate that suppression of IkappaBalpha expression is essential for JNK-mediated enhancement of Fas cytotoxicity.

TGF-beta-induced matrix proteins inhibit p42/44 MAPK and JNK activation and suppress TNF-mediated IkappaBalpha degradation and NF-kappaB nuclear translocation in L929 fibroblasts.

The role of transforming growth factor beta1 (TGF-beta1)-induced extracellular matrix proteins in the modulation of cellular response to the cytotoxic effect of tumor necrosis factor (TNF) or Fas ligand was investigated. Murine L929 fibroblasts were prestimulated with or without TGF-beta1 for 1-24 h and the resulting extracellular protein matrices were prepared. Unstimulated control L929 cells were then cultured on these matrices. Compared to control matrix-stimulated L929 cells, the TGF-beta1 matrix-stimulated cells resisted TNF killing in the presence of actinomycin D (ActD), but became more susceptible to killing by anti-Fas antibodies/ActD. The induced TNF resistance is independent of the NF-kappaB antiapoptotic effect. For example, exposure of TGF-beta1 matrix-stimulated L929 cells to TNF failed to result in IkappaBalpha degradation and NF-kappaB nuclear translocation or activation. Also, control matrix stimulated the activation of p42/44 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in L929 cells, whereas TGF-beta1 matrix suppressed the activation. Nonetheless, in response to TNF, JNK activation was restored in the TGF-beta1 matrix-stimulated cells. By metabolic labeling, ammonium sulfate precipitation and N-terminal amino acid microsequencing, TGF-beta1 was shown to induce a novel matrix protein of 46 kDa (p46) from L929 cells. Adsorption of p46 by peptide antibodies against its N-terminus removed the TGF-beta1 matrix protein-mediated protection against TNF/ActD cytotoxicity and its enhancement of anti-Fas/ActD killing, indicating that p46 is responsible for these effects. Immunostaining of L929 cells revealed that the antibodies were bound to a membrane protein of 100 kDa (p100). Thus, the matrix p46 is likely derived from the released membrane p100.

IkappaBalpha is essential for maintaining basal c-Jun N-terminal kinase (JNK) activation and regulating JNK-mediated resistance to tumor necrosis factor cytotoxicity in L929 cells.

Early activation of c-Jun N-terminal kinase (JNK) is believed to block apoptosis in response to death signals such as tumor necrosis factor (TNF). Brief exposure of murine L929 fibroblasts to anisomycin for 1 hr to activate JNK resulted in resistance to TNF killing. TNF rapidly induced cytoplasmic shrinkage in control cells, but not in the anisomycin-pretreated L929 cells. However, the induced TNF resistance was suppressed in the L929 cells which were engineered to stably inhibit IkappaBalpha protein expression by antisense mRNA ( approximately 80% reduction in protein expression). No constitutive NF-kappaB nuclear translocation and increased TNF resistance were found in these IkappaBalpha antisense cells. Notably, these cells had a significantly reduced basal level of JNK activation (50-70%), compared to vector control cells. Furthermore, brief exposure of L929 cells to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), resulted in resistance to TNF killing, probably due to preconsumption of caspases by wortmannin. Nonetheless, wortmannin-induced TNF resistance was suppressed in the IkappaBalpha antisense cells. Thus, these observations indicate that IkappaBalpha is essential for maintaining the basal level of JNK activation and regulating the JNK-induced TNF resistance.

Effects of removing the negatively charged N-terminal region of the salivary acidic proline-rich proteins by human leucocyte elastase.

Human leucocyte elastase from inflammatory gingival crevicular exudates (gingival crevicular fluid) contacts saliva and saliva-coated tooth surfaces coronal to the gingival margin. Major components of saliva are the salivary acidic proline-rich proteins (PRPs). These acidic PRPs, via the numerous negatively charged amino acid residues located predominantly within their amino-terminal region, bind to the hydroxyapatite mineral of the tooth surface and become part of the salivary pellicle. Thus the potential for human leucocyte elastase-mediated removal of the negatively charged amino-terminal region of acidic PRP variants (PRP-1, PRP-2, PRP-3, PRP-4, PIF-s and PIF-f) was examined. It was determined that each of the acidic PRP variants was susceptible to fragmentation by human leucocyte elastase, in which the 16 amino-terminal segment was removed, leaving the respective residual fragment named as the transitional product (tr). The transitional products were termed PRP-1tr, PRP-2tr (PIF-str), PRP-3tr and PRP-4tr (PIF-ftr). Each of the residual transitional products of acidic PRP had an amino-terminal beginning with serine residue no. 17, determined by amino acid sequencing. When samples of human leucocyte elastase-treated acidic PRPs were placed on native polyacrylamide gels and electrophoresed, the respective transitional products moved more slowly than the parental acidic PRP molecules, reflecting the loss of a portion of the negatively charged section. In comparison to the acidic PRPs, the acidic PRP transitional products had markedly reduced binding to hydroxyapatite. The transitional products were resistant to further enzymatic digestion as a function of increased incubation time and appeared to exert an antihuman leucocyte elastase effect. However, when increased concentrations of human leucocyte elastase were incubated with the acidic PRP, a more extensive digestion occurred, leaving a residual peptide with an amino-terminal beginning with alanine residue no. 44. Interestingly, intact acidic PRPs if prebound to hydroxyapatite particles, resisted digestion by human leucocyte elastase. In summary, human leucocyte elastase was capable of digesting fluid-phase (unbound) acidic PRP in a manner that eliminated part of their negatively charged region, which subsequently reduced their binding to hydroxyapatite. High concentrations of human leucocyte elastase, arriving from inflammatory gingival crevicular exudates, may interrupt the normal binding of fluid-phase acidic PRPs to hydroxyapatite.

Biomass conversion to mixed alcohol fuels using the MixAlco process.

The MixAlco process is a patented technology that converts any biodegradable material (e.g., sorted municipal solid waste, sewage sludge, industrial biosludge, manure, agricultural residues, energy crops) into mixed alcohol fuels containing predominantly 2-propanol, but also higher alcohols up to 7-tridecanol. The feedstock is treated with lime to increase its digestibility. Then, it is fed to a fermentor in which a mixed culture of acid-forming microorganisms produces carboxylic acids. Calcium carbonate is added to the fermentor to neutralize the acids to their corresponding carboxylate salt. The dilute (approximately 3%) carboxylate salts are concentrated to 19% using an amine solvent that selectively extracts water. Drying is completed using multi-effect evaporators. Finally, the dry salts are thermally converted to ketones which subsequently are hydrogenated to alcohols. All the steps in the MixAlco process have been proven at the laboratory scale. A techno-economic model of the process indicates that with the tipping fees available in New York (126 dollars/dry tonne), mixed alcohol fuels may be sold for 0.04 dollars/L (0.16 dollars/gal) with a 60% return on investment (ROI). With the average tipping fee in the United States rates (63 dollars/dry tonne), mixed alcohol fuels may be sold for 0.18 dollars/L (0.69 dollars/gal) with a 15% ROI. In the case of sugarcane bagasse, which may be obtained for about 26 dollars/dry ton, mixed alcohol fuels may be sold for 0.29 dollars/L (1.09 dollars/gal) with a 15% ROI.

p53 overexpression and downregulation of inter-alpha-inhibitor are associated with hyaluronidase enhancement of TNF cytotoxicity in L929 fibroblasts.

Degradation of extracellular matrix by hyaluronidase increases murine L929 cell sensitivity to tumor necrosis factor (TNF) cytotoxicity. Seeding and culturing L929 cells onto the matrix of serum fetuin and the hyaluronate-binding inter-alpha-inhibitor resulted in inhibition of hyaluronidase-enhanced TNF killing, suggesting that the release of these proteins from hyaluronidase-degraded matrix confers cellular TNF susceptibility. Metabolic labeling studies showed that hyaluronidase mediated de novo protein synthesis and down regulated several proteins in L929 cells. Specifically, hyaluronidase upregulated p53 protein expression (>200%) but down regulated a p85 inter-alpha-inhibitor-like protein (>90%) in L929 cells, whereas it had no effect on the protein levels of ICH-1, Bcl-xL, Bcl-2, Fas ligand, CAS (cellular apoptosis susceptible protein), TIAR (an RNA-binding protein) and alpha-tubulin. Conceivably, hyaluronidase enhancement of TNF sensitivity in L929 cells is p53-dependent and the matrix inter-alpha-inhibitor contributes a protective role against TNF cytotoxicity.

Transforming growth factor-beta protection of cancer cells against tumor necrosis factor cytotoxicity is counteracted by hyaluronidase (review).

Numerous cancer cells, when exposed to transforming growth factor beta (TGF-beta), become resistant to tumor necrosis factor (TNF) cytotoxicity. Pretreatment of L929 fibroblasts, for example, with TGF-beta isoforms (beta 1, beta 2 and beta 3) for at least 0.5-1 h results in resistance to TNF killing. TGF-beta 1 mediates the following sequential events in L929 cells: i) rapid induction of protein tyrosine-phosphorylation (< 30 min), ii) stimulation of protective protein synthesis and acquisition of TNF resistance (approximately 0.5-1 h), and iii) suppression of I kappa B-alpha expression (1-2 h). Two protective proteins induced by TGF-beta 1 are a 46 kDa extracellular matrix TNF-resistance triggering (TRT) protein and a putative transmembrane anti-apoptotic adhesion protein TIF2 (containing and RGD motif in the extracellular region). Both proteins enable L929 cells to resist TNF killing. Notably, testicular hyaluronidase increases TNF sensitivity in several types of cancer cells, counteracts TGF-beta-mediated TNF-resistance, and suppresses TGF-beta 1 gene expression in L929 cells in a serum-dependent manner. Moreover, hyaluronidase antagonizes TGF-beta-mediated inhibition of epithelial cell growth. Both TGF-beta and hyaluronidase are essential for the progression and invasiveness of breast, prostate and other cancers. Conceivably, a stage-dependent expression, as well as a balanced production, of these proteins is essential for cancer development and self protection against TNF cytotoxicity.