Instanton approach to large N Harish-Chandra-Itzykson-Zuber integrals.

We reconsider the large N asymptotics of Harish-Chandra-Itzykson-Zuber integrals. We provide, using Dyson's Brownian motion and the method of instantons, an alternative, transparent derivation of the Matytsin formalism for the unitary case. Our method is easily generalized to the orthogonal and symplectic ensembles. We obtain an explicit solution of Matytsin's equations in the case of Wigner matrices, as well as a general expansion method in the dilute limit, when the spectrum of eigenvalues spreads over very wide regions.

A PPM-family protein phosphatase from the thermoacidophile Thermoplasma volcanium hydrolyzes protein-bound phosphotyrosine.

The genomes of virtually all free-living archaeons encode one or more deduced protein-serine/threonine/tyrosine kinases belonging to the so-called eukaryotic protein kinase superfamily. However, the distribution of their cognate protein-serine/threonine/phosphatases displays a mosaic pattern. Thermoplasma volcanium is unique among the Archaea inasmuch as it is the sole archaeon whose complement of deduced phosphoprotein phosphatases includes a member of the PPM-family of protein-serine/threonine phosphatases--a family that originated in the Eucarya. A recombinant version of this protein, TvnPPM, exhibited protein-tyrosine phosphatase in addition to its predicted protein-serine/threonine phosphatase activity in vitro. TvnPPM is the fourth member of the PPM-family shown to exhibit such dual-specific capability, suggesting that the ancestral versions of this enzyme exhibited broad substrate specificity. Unlike most other archaeons, the genome of T. volcanium lacks open reading frames encoding stereotypical protein-tyrosine phosphatases. Hence, the dual-specificity of TvnPPM may account for its seemingly aberrant presence in an archaeon.

The protein phosphatases of Synechocystis sp. strain PCC 6803: open reading frames sll1033 and sll1387 encode enzymes that exhibit both protein-serine and protein-tyrosine phosphatase activity in vitro.

The open reading frames (ORFs) encoding two potential protein-serine/threonine phosphatases from the cyanobacterium Synechocystis sp. strain PCC 6803 were cloned and their protein products expressed in Escherichia coli cells. The product of ORF sll1033, SynPPM3, is a homologue of the PPM family of protein-serine/threonine phosphatases found in all eukaryotes as well as many members of the Bacteria. Surprisingly, the recombinant protein phosphatase dephosphorylated phosphotyrosine- as well as phosphoserine-containing proteins in vitro. While kinetic analyses indicate that the enzyme was more efficient at dephosphorylating the latter, replacement of Asp608 by asparagine enhanced activity toward a phosphotyrosine-containing protein fourfold. The product of ORF sll1387, SynPPP1, is the sole homolog of the PPP family of protein phosphatases encoded by the genome of Synechocystis sp. strain PCC 6803. Like many other bacterial PPPs, the enzyme dephosphorylated phosphoserine- and phosphotyrosine-containing proteins with comparable efficiencies. However, while previously described PPPs from prokaryotic organisms required the addition of exogenous metal ion cofactors, such as Mg2+ or Mn2+, for activity, recombinantly produced SynPPP1 displayed near-maximal activity in the absence of added metals. Inductively coupled plasma mass spectrometry indicated that recombinant SynPPP1 contained significant quantities, 0.32 to 0.44 mol/mole total, of Mg and Mn. In this respect, the cyanobacterial enzyme resembled eukaryotic members of the PPP family, which are metalloproteins. mRNA encoding SynPPP1 or SynPPM3 could be detected in cells grown under many, but not all, environmental conditions.

A phosphoprotein from the archaeon Sulfolobus solfataricus with protein-serine/threonine kinase activity.

Sulfolobus solfataricus contains a membrane-associated protein kinase activity that displays a strong preference for threonine as the phospho-acceptor amino acid residue. When a partially purified detergent extract of the membrane fraction from the archaeon S. solfataricus that had been enriched for this activity was incubated with [gamma-(32)P]ATP, radiolabeled phosphate was incorporated into roughly a dozen polypeptides, several of which contained phosphothreonine. One of the phosphothreonine-containing proteins was identified by mass peptide profiling as the product of open reading frame [ORF] sso0469. Inspection of the DNA-derived amino acid sequence of the predicted protein product of ORF sso0469 revealed the presence of sequence characteristics faintly reminiscent of the "eukaryotic" protein kinase superfamily. ORF sso0469 therefore was cloned, and its polypeptide product was expressed in Escherichia coli. The recombinant protein formed insoluble aggregates that could be dispersed using urea or detergents. The solubilized polypeptide phosphorylated several exogenous proteins in vitro, including casein, myelin basic protein, and bovine serum albumin. Mutagenic alteration of amino acids predicted to be essential for catalytic activity abolished or severely reduced catalytic activity. Phosphorylation of exogenous substrates took place on serine and, occasionally, threonine. This new archaeal protein kinase displayed no catalytic activity when GTP was substituted for ATP as the phospho-donor substrate, while Mn(2+) was the preferred cofactor.

Phosphoprotein with phosphoglycerate mutase activity from the archaeon Sulfolobus solfataricus.

When soluble extracts of the extreme acidothermophilic archaeon Sulfolobus solfataricus were incubated with [gamma-(32)P]ATP, several proteins were radiolabeled. One of the more prominent of these, which migrated with a mass of approximately 46 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was purified by column chromatography and SDS-PAGE and subjected to amino acid sequence analysis via both the Edman technique and mass spectroscopy. The best match to the partial sequence obtained was the potential polypeptide product of open reading frame sso0417, whose DNA-derived amino acid sequence displayed many features reminiscent of the 2,3-diphosphoglycerate-independent phosphoglycerate (PGA) mutases [iPGMs]. Open reading frame sso0417 was therefore cloned, and its protein product was expressed in Escherichia coli. Assays of its catalytic capabilities revealed that the protein was a moderately effective PGA mutase that also exhibited low levels of phosphohydrolase activity. PGA mutase activity was dependent upon the presence of divalent metal ions such as Co(2+) or Mn(2+). The recombinant protein underwent autophosphorylation when incubated with either [gamma-(32)P]ATP or [gamma-(32)P]GTP. The site of phosphorylation was identified as Ser(59), which corresponds to the catalytically essential serine residue in bacterial and eucaryal iPGMs. The phosphoenzyme intermediate behaved in a chemically and kinetically competent manner. Incubation of the (32)P-labeled phosphoenzyme with 3-PGA resulted in the disappearance of radioactive phosphate and the concomitant appearance of (32)P-labeled PGA at rates comparable to those measured in steady-state assays of PGA mutase activity.

Leverage effect in financial markets: the retarded volatility model.

We investigate quantitatively the so-called "leverage effect," which corresponds to a negative correlation between past returns and future volatility. For individual stocks this correlation is moderate and decays over 50 days, while for stock indices it is much stronger but decays faster. For individual stocks the magnitude of this correlation has a universal value that can be rationalized in terms of a new "retarded" model which interpolates between a purely additive and a purely multiplicative stochastic process. For stock indices a specific amplification phenomenon seems to be necessary to account for the observed amplitude of the effect.

Comparative pathogenesis of infection of pigs with hepatitis E viruses recovered from a pig and a human.

Specific-pathogen-free pigs were inoculated with one of two hepatitis E viruses (HEV) (one recovered from a pig and the other from a human) to study the relative pathogenesis of the two viruses in swine. Fifty-four pigs were randomly assigned to three groups. Seventeen pigs in group 1 served as uninoculated controls, 18 pigs in group 2 were intravenously inoculated with the swine HEV recovered from a pig in the United States, and 19 pigs in group 3 were intravenously inoculated with the US-2 strain of human HEV recovered from a hepatitis patient in the United States. Two to four pigs from each group were necropsied at 3, 7, 14, 20, 27, or 55 days postinoculation (DPI). Evidence of clinical disease or elevation of liver enzymes or bilirubin was not found in pigs from any of the three groups. Enlarged hepatic and mesenteric lymph nodes were observed in both HEV-inoculated groups. Multifocal lymphoplasmacytic hepatitis was observed in 9 of 17, 15 of 18, and 16 of 19 pigs in groups 1 to 3, respectively. Focal hepatocellular necrosis was observed in 5 of 17, 10 of 18, and 13 of 19 pigs in groups 1 to 3, respectively. Hepatitis lesions were very mild in group 1 pigs, mild to moderate in group 2 pigs, and moderate to severe in group 3 pigs. Hepatic inflammation and hepatocellular necrosis peaked in severity at 20 DPI and were still moderately severe at 55 DPI in the group inoculated with human HEV. Hepatitis lesions were absent or nearly resolved by 55 DPI in the swine-HEV-inoculated pigs. All HEV-inoculated pigs seroconverted to anti-HEV immunoglobulin G. HEV RNA was detected by reverse transcriptase PCR in feces, liver tissue, and bile of pigs in both HEV-inoculated groups from 3 to 27 DPI. Based on evaluation of microscopic lesions, the US-2 strain of human HEV induced more severe and persistent hepatic lesions in pigs than did swine HEV. Pig livers or cells from the livers of HEV-infected pigs may represent a risk for transmission of HEV from pigs to human xenograft recipients. Since HEV was shed in the feces of infected pigs, exposure to feces from infected pigs represents a risk for transmission of HEV, and pigs should be considered a reservoir for HEV.

Characterization of a 12-kilodalton rhodanese encoded by glpE of Escherichia coli and its interaction with thioredoxin.

Rhodaneses catalyze the transfer of the sulfane sulfur from thiosulfate or thiosulfonates to thiophilic acceptors such as cyanide and dithiols. In this work, we define for the first time the gene, and hence the amino acid sequence, of a 12-kDa rhodanese from Escherichia coli. Well-characterized rhodaneses are comprised of two structurally similar ca. 15-kDa domains. Hence, it is thought that duplication of an ancestral rhodanese gene gave rise to the genes that encode the two-domain rhodaneses. The glpE gene, a member of the sn-glycerol 3-phosphate (glp) regulon of E. coli, encodes the 12-kDa rhodanese. As for other characterized rhodaneses, kinetic analysis revealed that catalysis by purified GlpE occurs by way of an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. The K(m)s for SSO(3)(2-) and CN(-) were 78 and 17 mM, respectively. The apparent molecular mass of GlpE under nondenaturing conditions was 22.5 kDa, indicating that GlpE functions as a dimer. GlpE exhibited a k(cat) of 230 s(-1). Thioredoxin 1 from E. coli, a small multifunctional dithiol protein, served as a sulfur acceptor substrate for GlpE with an apparent K(m) of 34 microM when thiosulfate was near its K(m), suggesting that thioredoxin 1 or related dithiol proteins could be physiological substrates for sulfurtransferases. The overall degree of amino acid sequence identity between GlpE and the active-site domain of mammalian rhodaneses is limited ( approximately 17%). This work is significant because it begins to reveal the variation in amino acid sequences present in the sulfurtransferases. GlpE is the first among the 41 proteins in COG0607 (rhodanese-related sulfurtransferases) of the database Clusters of Orthologous Groups of proteins (http://www.ncbi.nlm.nih.gov/COG/) for which sulfurtransferase activity has been confirmed.

Immune reactions in classical Hodgkin's lymphoma.

The immune reaction in classical Hodgkin's lymphoma (HL) can be separated into an inflammatory response in the involved tissues and a generalized immune response in the patient. The local immune reaction in HL is by far the most prominent among all tumors, with the exception of so called T-cell-rich B-cell lymphoma, a subtype of large-cell B-cell lymphoma. The general immune response in patients with HL is best described as an acquired cellular immune deficiency, most likely a result of the presence of tumor, although some data in the literature suggest a preexisting immune deficiency. The cellular origin of Reed-Sternberg (RS) cells in classical and nodular lymphocyte-predominant HL is discussed elsewhere. RS cells and their mononuclear variants can be considered as clones of neoplastic B cells that, by secreting potent cytokines/chemokines, not only cause the symptoms of HL but also promote their own growth and evade immune surveillance. The characteristic histologic features of HL--the prominent inflammatory response, the influx of eosinophils, and the presence of fibrosis and sclerosis--may also result from the expression of a range of surface molecules and the production of cytokines and chemokines by RS cells.

An insect homolog of the vertebrate very low density lipoprotein receptor mediates endocytosis of lipophorins.

A novel member of the low density lipoprotein (LDL) receptor family was identified, which is expressed in locust oocytes, fat body, brain, and midgut. This receptor appeared to be a homolog of the mammalian very low density lipoprotein receptor as it contains eight cysteine-rich repeats in its putative ligand-binding domain. When transiently expressed in COS-7 or stably expressed in LDL receptor-deficient CHO cells, the receptor mediates endocytic uptake of high density lipophorin (HDLp), an abundant lipoprotein in the circulatory compartment of insects. Moreover, in the latter cell line, we demonstrated that an excess of unlabeled HDLp competed with fluorescent labeled HDLp for uptake whereas an excess of human LDL did not affect uptake. Expression of the receptor mRNA in fat body cells is down-regulated during adult development, which is consistent with the previously reported down-regulation of receptor-mediated endocytosis of lipophorins in fat body tissue (Dantuma, N. P., M.A.P. Pijnenburg, J. H. B. Diederen, and D. J. Van der Horst. 1997. J. Lipid Res. 38: 254-265). The expression of this receptor in various tissues that internalize circulating lipophorins and its capability to mediate endocytosis of HDLp indicate that this novel member of the LDL receptor family may function as an endocytic lipophorin receptor in vivo.

Immune escape mechanisms in Hodgkin's disease.

BACKGROUND: The nodular sclerosis and mixed cellularity subtypes of Hodgkin's disease are histologically characterised by a small population of neoplastic cells, the so-called Reed-Sternberg cells and their mononuclear variants (RS cells) and an extensive admixture of other cell types including lymphocytes, plasma cells, eosinophils, and histiocytes. The nature of this infiltrate is largely known, but the mechanisms and functional effects are not. The small lymphocytes immediately surrounding the RS cells are mostly CD4+ T cells that express early activation markers. The absence of prominent specific cytotoxic T cell or natural killer (NK) cell populations seems to argue against a Th1-type response, whereas the sometimes prominent admixture of plasma cells and eosinophils is suggestive of a Th2-type response. Enrichment of the CD4 T-cell population may result from selective influx of CD4 T cells or from selective depletion of CD8 and NK cells. RESULTS AND DISCUSSION: The T cells surrounding RS cells have an immuno-phenotype and cytokine production capability consistent with a Th2-type response. RS cells express several members of the TNF receptor family such as the FAS ligand (CD95L) that may induce apoptosis of activated, FAS expressing, CD8+ T cells and NK cells. The RS cells also produce TGF beta and interleukin-10 that may downmodulate the Th1 response. In addition, the Reed-Sternberg cells produce the chemokine TARC that could lead to the specific attraction of a Th2 T-cell subset. CONCLUSION: RS cells have several mechanisms that may allow it to escape an effective immune response. The relative contributions of each of these and other potential mechanisms are not yet known.

Organotin-induced apoptosis as observed in vitro is not relevant for induction of thymus atrophy at antiproliferative doses.

The organotin compounds di-n-butyltin dichloride (DBTC) and tri-n-butyltin chloride (TBTC) selectively cause thymus atrophy. Previously, DBTC and TBTC were shown to inhibit proliferation of immature thymocytes, but other studies demonstrated that TBTC but not DBTC increased apoptosis in vitro and also in vivo. In this study, we examined whether apoptosis is increased in vitro by DBTC and TBTC at various concentrations and periods of incubation and whether apoptosis is involved in the induction of thymus atrophy at selective antiproliferative doses. In vitro, DBTC or TBTC at a concentration of 3 microM significantly increased DNA fragmentation in freshly isolated rat thymocytes after preincubation for 10 min followed by a 22-hr culture period. After continuous exposure for 22 hr, apoptosis was observed to be optimum at 1 microM TBTC and 0.3 microM DBTC and lower or absent at higher concentrations. Apparently, apoptosis induced by organotins in vitro depends on both duration and concentration of exposure. Selective antiproliferative doses of DBTC nor TBTC increased apoptosis on day 1 or 2 after single oral exposure. In contrast, the corticosteroid dexamethasone caused a depletion of both small and large thymocytes and a marked increase of apoptosis on both days 1 and 2 after dosing. Thus, although apoptosis is involved in the in vitro cytotoxic effects of both organotin compounds, it seems not involved in thymus atrophy at a dose that selectively inhibits immature thymocyte proliferation.