EpCAM regulates cell cycle progression via control of cyclin D1 expression.

The epithelial cell adhesion molecule (EpCAM) is an integral transmembrane protein that is frequently overexpressed in embryonic stem cells, tissue progenitors, carcinomas and cancer-initiating cells. In cancer cells, expression of EpCAM is associated with enhanced proliferation and upregulation of target genes including c-myc. However, the exact molecular mechanisms underlying the observed EpCAM-dependent cell proliferation remained unexplored. Here, we show that EpCAM directly affects cell cycle progression via its capacity to regulate the expression of cyclin D1 at the transcriptional level and depending on the direct interaction partner FHL2 (four-and-a-half LIM domains protein 2). As a result, downstream events such as phosphorylation of the retinoblastoma protein (Rb) and expression of cyclins E and A are similarly affected. In vivo, EpCAM expression strength and pattern are both positively correlated with the proliferation marker Ki67, high expression and nuclear localisation of cyclin D1, and Rb phosphorylation. Thus, EpCAM enhances cell cycle progression via the classical cyclin-regulated pathway.

Phosphorus-enriched hemodialysis for the treatment of patients with severe methanol intoxication.

Severe methanol poisoning requires treatment with prolonged and intensive hemodialytic therapy. Such treatment can engender either the de novo development of hypophosphatemia or the worsening of pre-existing hypophosphatemia. Phosphorus-enriched hemodialysis therapy can prevent the occurrence of this complication. We report three patients with severe methanol poisoning who were treated with phosphorus-enriched hemodialysis. Prevention or treatment of hypophosphatemia was successfully achieved with this dialytic technique.

Methylation of Sm proteins by a complex containing PRMT5 and the putative U snRNP assembly factor pICln.

Seven Sm proteins, termed B/B', D1, D2, D3, E, F, and G, assemble in an ordered manner onto U snRNAs to form the Sm core of the spliceosomal snRNPs U1, U2, U4/U6, and U5. The survival of motor neuron (SMN) protein binds to Sm proteins and mediates in the context of a macromolecular (SMN-) complex the assembly of the Sm core. Binding of SMN to Sm proteins is enhanced by modification of specific arginine residues in the Sm proteins D1 and D3 to symmetrical dimethylarginines (sDMAs), suggesting that assembly might be regulated at the posttranslational level. Here we provide evidence that the previously described pICln-complex, consisting of Sm proteins, the methyltransferase PRMT5, pICln, and two novel factors, catalyzes the sDMA modification of Sm proteins. In vitro studies further revealed that the pICln complex inhibits the spontaneous assembly of Sm proteins onto a U snRNA. This effect is mediated by pICln via its binding to the Sm fold of Sm proteins, thereby preventing specific interactions between Sm proteins required for the formation of the Sm core. Our data suggest that the pICln complex regulates an early step in the assembly of U snRNPs, possibly the transfer of Sm proteins to the SMN-complex.

Cloning and characterization of a second laccase gene from the lignin-degrading basidiomycete Pycnoporus cinnabarinus.

The gene lcc3-2 encoding a second laccase of the white-rot fungus Pycnoporus cinnabarinus has been cloned, sequenced, and characterized. The isolated gene consists of 2840bp, with the coding region interrupted by ten introns and flanked by an upstream region in which putative CAAT and TATA boxes were identified. The cDNA of lcc3-2 contains an open reading frame of 1563bp. The deduced mature laccase protein consisted of 498 amino acids and was preceded by a signal peptide of 23 amino acids. The sequence of lcc3-2 reveals 73% similarity on the protein level to the previously characterized lcc3-1. The new laccase gene shares highest similarity to lcc1 from Trametes villosa (75%), and lcc2 from the unidentified basidiomycete CECT 20197 (75%). The calculated isoelectric point (pI) of 6.1 for the gene product LCC3-2 was in good agreement with the experimentally determined pI of a laccase secreted by P. cinnabarinus grown on cellulose. Transcription analysis using competitive reverse transcription (RT)-PCR showed that lcc3-2 was expressed in glucose and cellulose containing cultures. However, in contrast to lcc3-1, lcc3-2 transcription was not increased in response to 2,5-xylidine.

Novel interaction between laccase and cellobiose dehydrogenase during pigment synthesis in the white rot fungus Pycnoporus cinnabarinus.

When glucose is the carbon source, the white rot fungus Pycnoporus cinnabarinus produces a characteristic red pigment, cinnabarinic acid, which is formed by laccase-catalyzed oxidation of the precursor 3-hydroxyanthranilic acid. When P. cinnabarinus was grown on media containing cellobiose or cellulose as the carbon source, the amount of cinnabarinic acid that accumulated was reduced or, in the case of cellulose, no cinnabarinic acid accumulated. Cellobiose-dependent quinone reducing enzymes, the cellobiose dehydrogenases (CDHs), inhibited the redox interaction between laccase and 3-hydroxyanthranilic acid. Two distinct proteins were purified from cellulose-grown cultures of P. cinnabarinus; these proteins were designated CDH I and CDH II. CDH I and CDH II were both monomeric proteins and had apparent molecular weights of about 81,000 and 101,000, respectively, as determined by both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI values were approximately 5.9 for CDH I and 3.8 for CDH II. Both CDHs used several known CDH substrates as electron acceptors and specifically adsorbed to cellulose. Only CDH II could reduce cytochrome c. The optimum pH values for CDH I and CDH II were 5.5 and 4.5, respectively. In in vitro experiments, both enzymes inhibited laccase-mediated formation of cinnabarinic acid. Oxidation intermediates of 3-hydroxyanthranilic acid served as endogenous electron acceptors for the two CDHs from P. cinnabarinus. These results demonstrated that in the presence of a suitable cellulose-derived electron donor, CDHs can regenerate fungal metabolites oxidized by laccase, and they also supported the hypothesis that CDHs act as links between cellulolytic and ligninolytic pathways.

Wear of nickel-titanium lightspeed instruments evaluated by scanning electron microscopy.

Used rotary nickel-titanium instruments require frequent replacing. This laboratory study evaluated defects of Lightspeed cutting tips before and after usage. The instruments were fixed into custom-made holders, the cutting heads photographed in a scanning electron microscope at x120 to x400 magnification at preset points around the cutting tip (90, 180, 270 and 360 degrees) and head-on. Instrument sizes 20 to 32.5, 35 to 60, and 65 to 100 were used in 9, 18, and 36 canals, respectively, and autoclaved after shaping every third root canal. The used instruments were cleaned and then reexamined in a scanning electron microscope as before. The presence of 11 types of conditions was scored from the pre- and postusage photographs. No instruments fractured during the test, but all the cutting heads had one or more imperfections, even before usage. The presence of debris, pitting, and metal strips changed significantly. Imperfections were found on new and used Lightspeed cutting heads, indicating the general difficulty in machining defect-free nickel-titanium rotary instruments. However, high quality should remain a goal to improve instrument efficiency.

Molecular analysis of a laccase gene from the white rot fungus Pycnoporus cinnabarinus.

It was recently shown that the white rot basidiomycete Pycnoporus cinnabarinus secretes an unusual set of phenoloxidases when it is grown under conditions that stimulate ligninolysis (C. Eggert, U. Temp, and K.-E. L. Eriksson, Appl. Environ. Microbiol. 62:1151-1158, 1996). In this report we describe the results of a cloning and structural analysis of the laccase-encoding gene (lcc3-1) expressed by P. cinnabarinus during growth under xylidine-induced conditions. The coding region of the genomic laccase sequence, which is preceded by the eukaryotic promoter elements TATA and CAATA, spans more than 2,390 bp. The corresponding laccase cDNA was identical to the genomic sequence except for 10 introns that were 50 to 60 bp long. A sequence analysis indicated that the P. cinnabarinus lcc3-1 product has a Phe residue at a position likely to influence the reduction-oxidation potential of the enzyme's type 1 copper center. The P. cinnabarinus lcc3-1 sequence was most similar to the sequence encoding a laccase from Coriolus hirsutus (level of similarity, 84%).

A small-scale method for screening of lignin-degrading microorganisms.

A new method to facilitate rapid screening of lignin-degrading microorganisms was developed. Fungal strains are cultivated in tissue culture plates containing C-ring-labeled dehydrogenation polymerizate (DHP) (synthetic lignin). Evolved CO(2) is trapped in barium-saturated filter paper and is detected by exposing the paper to X-ray film. Analysis of the autoradiograms, carried out by density measurement with an image analysis program, allows for a semiquantitative estimation of the amount of CO(2) evolved. The method is especially useful for screening for new, powerful lignin-degrading strains in both man-made and natural environments. It eliminates the need for special equipment for their cultivation and trapping of CO(2) as well as laborious sample analysis. The method has in this study been used to test three novel fungal isolates and a laccaseless mutant of the basidiomycete Pycnoporus cinnabarinus. Their ligninolytic capacities were compared with those of the potent lignin degrader Ceriporiopsis subvermispora.

Laccase-catalyzed formation of cinnabarinic acid is responsible for antibacterial activity of Pycnoporus cinnabarinus.

Concentrated culture fluid of the wood-rotting basidiomycete Pycnoporus cinnabarinus showed biological activity against a variety of bacterial strains. The maximal inhibitory effect was obtained for Gram-positive bacteria of the genus Streptococcus. In general, inhibition was higher for Gram-positive than Gram-negative bacteria. P. cinnabarinus produces the phenoxazinone derivative, cinnabarinic acid. This red pigment accumulates in sporocarps as well as in liquid cultures. As shown previously, laccase secreted by the fungus oxidizes the precursor 3-hydroxyanthranilic acid to cinnabarinic acid. The present study demonstrates that this reaction is necessary for the production of antibacterial compounds by the fungus. The biological activity of concentrated P. cinnabarinus culture fluid was nearly identical with that of cinnabarinic acid, synthesized by purified laccase in vitro.

[Functional electromyography analysis of radial replacement operation]. / Elektromyographische Funktionsanalyse der Radialisersatzoperation.

The aim of this electromyographic study was to evaluate the functional result of tendon transfers for radial palsy. Eighteen patients were compared to 16 healthy volunteers. The intramuscular emg of four muscles was recorded in parallel during extension-flexion movements of the wrist and fingers. The data were stored on FM tape and evaluated off-line. Contrary to expectations, transferred flexor muscles revealed only part of their overall emg activity during extension. Therefore, an "extensor quota" (EQ) was calculated for each muscle. It reflects its emg activity during extension as part of its entire emg activity. In non-transferred flexors EQ was low (11-27%). High EQ values (69-91%) were seen in genuine extensors of healthy persons. In only 6 of 18 patients did transferred flexors show an EQ equivalent to genuine extensors (> or = 69%). Best functional results were seen in transferred flexor carpi ulnaris (median EQ 68%). The EQ of transferred pronator teres was low (median 18%) and minor to flexor carpi ulnaris in each patient (P < 0.05). It is concluded that in cases of radial palsy, pronator teres is not suitable for replacing extensor muscles. Superficial flexors of the fingers, however, work in synergy with extensors of the wrist. According to the la reflex connections involved, these muscles should be a good choice for regaining extension of the wrist.

Laccase is essential for lignin degradation by the white-rot fungus Pycnoporus cinnabarinus.

The white-rot fungus, Pycnoporus cinnabarinus, provides an excellent model organism to elucidate the controversial role of laccase in lignin degradation. P. cinnabarinus produces laccase in one isoform as the predominant phenoloxidase in ligninolytic cultures, and neither LiP nor MnP are secreted. Yet, P. cinnabarinus degrades lignin very efficiently. In the present work, we show that laccase-less mutants of P. cinnabarinus were greatly reduced in their ability to metabolize 14C ring-labeled DHP. However, 14CO2 evolution in these mutant cultures could be restored to levels comparable to those of the wild-type cultures by addition of purified P. cinnabarinus laccase. This clearly indicates that laccase is absolutely essential for lignin degradation by P. cinnabarinus.

Electromyographic study on the function of tendon transfers in radial palsy.

The aim of this electromyographic study was to evaluate the functional result of tendon transfers for radial palsy. Eighteen patients were compared to 16 healthy volunteers. The intramuscular emg of four muscles was recorded in parallel during extension-flexion movements of the wrist and fingers. The data were stored on FM tape and evaluated off-line. Contrary to expectations, transferred flexor muscles revealed only part of their overall emg activity during extension. Therefore, an "extensor quota" (EQ) was calculated for each muscle. It reflects its emg activity during extension as part of its entire emg activity. In non-transferred flexors EQ was low (11-27 %). High EQ values (69-91 %) were seen in genuine extensors of healthy persons. In only 6 of 18 patients did transferred flexors show an EQ equivalent to genuine extensors (≥ 69 %). Best functional results were seen in transferred flexor carpi ulnaris (median EQ 68 %). The EQ of transferred pronator teres was low (median 18 %) and minor to flexor carpi ulnaris in each patient (P < 0.05). It is concluded that in cases of radial palsy, pronator teres is not suitable for replacing extensor muscles. Superficial flexors of the fingers, however, work in synergy with extensors of the wrist. According to the Ia reflex connections involved, these muscles should be a good choice for regaining extension of the wrist.

A fungal metabolite mediates degradation of non-phenolic lignin structures and synthetic lignin by laccase.

Lignin peroxidase is generally considered to be a primary catalyst for oxidative depolymerization of lignin by white-rot fungi. However, some white-rot fungi lack lignin peroxidase. Instead, many produce laccase, even though the redox potentials of known laccases are too low to directly oxidize the non-phenolic components of lignin. Pycnoporus cinnabarinus is one example of a laccase-producing fungus that degrades lignin very efficiently. To overcome the redox potential barrier, P. cinnabarinus produces a metabolite, 3-hydroxyanthranilate that can mediate the oxidation of how non-phenolic substrates by laccase. This is the first description of how laccase might function in a biological system for the complete depolymerization of lignin.

The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase.

The white rot fungus Pycnoporus cinnabarinus was characterized with respect to its set of extracellular phenoloxidases. Laccase was produced as the predominant extracellular phenoloxidase in conjunction with low amounts of an unusual peroxidase. Neither lignin peroxidase nor manganese peroxidase was detected. Laccase was produced constitutively during primary metabolism. Addition of the most effective inducer, 2,5-xylidine, enhanced laccase production ninefold without altering the isoenzyme pattern of the enzyme. Laccase purified to apparent homogeneity was a single polypeptide having a molecular mass of approximately 81,000 Da, as determined by calibrated gel filtration chromatography, and a carbohydrate content of 9%. The enzyme displayed an unusual behavior on isoelectric focusing gels; the activity was split into one major band (pI, 3.7) and several minor bands of decreasing intensity which appeared at regular, closely spaced intervals toward the alkaline end of the gel. Repeated electrophoresis of the major band under identical conditions produced the same pattern, suggesting that the laccase was secreted as a single acidic isoform with a pI of about 3.7 and that the multiband pattern was an artifact produced by electrophoresis. This appeared to be confirmed by N-terminal amino acid sequencing of the purified enzyme, which yielded a single sequence for the first 21 residues. Spectroscopic analysis indicated a typical laccase active site in the P. cinnabarinus enzyme since all three typical Cu(II)-type centers were identified. Substrate specificity and inhibitor studies also indicated the enzyme to be a typical fungal laccase. The N-terminal amino acid sequence of the P. cinnabarinus laccase showed close homology to the N-terminal sequences determined for laccases from Trametes versicolor, Coriolus hirsutus, and an unidentified basidiomycete, PM1. The principal features of the P. cinnabarinus enzyme system, a single predominant laccase and a lack of lignin- or manganese-type peroxidase, make this organism an interesting model for further studies of possible alternative pathways of lignin degradation by white rot fungi.

Laccase-mediated formation of the phenoxazinone derivative, cinnabarinic acid.

The phenoxazinone chromophore occurs in a variety of biological systems, including numerous pigments and certain antibiotics. It also appears to form as part of a mechanism to protect mammalian tissue from oxidative damage. During cultivation of the basidiomycete, Pycnoporus cinnabarinus, a red pigment was observed to accumulate in the culture medium. It was identified as the phenoxazinone derivative, cinnabarinic acid (CA). Laccase was the predominant extracellular phenoloxidase activity in P. cinnabarinus cultures. In vitro studies showed that CA was formed after oxidation of the precursor, 3-hydroxyanthranilic acid (3-HAA), by laccases. Moreover, oxidative coupling of 3-HAA to form CA was also demonstrated for the mammalian counterpart of laccase, the blue copper oxidase, ceruloplasmin.

Degradability of chlorine-free bleachery effluent lignins by two fungi: effects on lignin subunit type and on polymer molecular weight.

A bleachery effluent from a sulfite process pulp mill, which was extracted with alkali and treated with oxygen and hydrogen peroxide (EOP), was treated with two fungi, Trametes versicolor and Stagonospora gigaspora. Trametes versicolor did not cause any depolymerization or degradation of effluent lignins but increased the amount of chromophores, whereas S. gigaspora depolymerized the EOP lignins and caused a substantial reduction in aromatic compounds. For both fungal treatments, CuO oxidation caused a decrease in the yield of the aldehydes within the vanillyl and p-hydroxy phenol families, which was faster than the rates of decrease in the yields of the corresponding acids and ketones. However, only S. gigaspora caused changes in the pattern of the 11 characteristic lignin phenols produced by CuO oxidation, reflecting a preferential metabolism of some phenolic precursors. This fungus decreased the yield of total vanillyl phenols (V), which contributed the bulk of the 11 lignin oxidation products, from 93% initially to 59%. As a consequence, coumaryl (C), syringyl (S), and p-hydroxy phenols (P) became relatively enriched to 1.2, 6.5, and 33%, respectively. The stability of EOP-lignin constituent subunits is S > P > C > V. The two fungi differed significantly in their level of enzyme activities. In effluent-free medium, the ratio of laccase to peroxidase was higher for T. versicolor than for S. gigaspora. The presence of EOP-lignins significantly increased this ratio. No lignin peroxidase was detected but manganese peroxidase and laccase were detected during degradation activities.