Immunization with tumor-associated epitopes fused to an endoplasmic reticulum translocation signal sequence affords protection against tumors with down-regulated expression of MHC and peptide transporters

Treatment of human cancers with an inherent antigen-processing defect due to a loss of peptide transporters (TAP-1 and TAP-2) and/or MHC class I antigen expression remains a considerable challenge. There is now an increasing realization that tumor cells with down-regulated expression of TAP and/or MHC class I antigens display strong resistance to cytotoxic T lymphocyte (CTL)mediated immune control, and often fail to respond to the conventional immunotherapeutic protocols based on active immunization with tumor-associated epitopes (TAE) or adoptive transfer of tumor-specific T cells, In the present study, we describe a novel approach based on immunization with either genetically modified tumor cells or naked DNA vectors encoding TAE fused to an endoplasmic reticulum (ER) signal sequence (ER-TAE) which affords protection against challenge by melanoma cells with down-regulated expression of TAP-1/2 and MHC class I antigens. In contrast, animals immunized with a vaccine based on TAE alone showed no protection against tumor challenge. Although MHC-peptide tetramer analysis showed a similar frequency of antigen-specific CTL in both ER-TAE- and TAE-immunized mice, functional analysis revealed that CTL activated following immunization with ER-TAE displayed significantly higher avidity for TAE when compared to animals immunized with the TAE alone, These observations provide a new strategy in anti-cancer vaccine design that allows activation of a highly effective and well-defined CTL response against tumors with down-regulated expression of TAP and MHC class I antigens.

Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions

It has been previously observed that the intrinsically weak variant GC donor sites, in order to be recognized by the U2-type spliceosome, possess strong consensus sequences maximized for base pair formation with U1 and U5/U6 snRNAs. However, variability in signal strength is a fundamental mechanism for splice site selection in alternative splicing. Here we report human alternative GC-AG introns (for the first time from any species), and show that while constitutive GC-AG introns do possess strong signals at their donor sites, a large subset of alternative GC-AG introns possess weak consensus sequences at their donor sites. Surprisingly, this subset of alternative isoforms shows strong consensus at acceptor exon positions 1 and 2. The improved consensus at the acceptor exon can facilitate a strong interaction with U5 snRNA, which tethers the two exons for ligation during the second step of splicing. Further, these isoforms nearly always possess alternative acceptor sites and always possess alternative acceptor sites and exhibit particularly weak polypyrimidine tracts characteristic of AG-dependent introns. The acceptor exon nucleotides are part of the consensus required for the U2AF(35)-mediated recognition of AG in such introns. Such improved consensus at acceptor exons is not found in either normal or alternative GT-AG introns having weak donor sites or weak polypyrimidine,tracts. The changes probably reflect mechanisms that allow GC-AG alternative intron isoforms to cope with two conflicting requirements, namely an apparent need for differential splice strength to direct the choice of alternative sites and a need for improved donor signals to compensate for the central mismatch base pair (C-A) in the RNA duplex of U1 snRNA and the pre-mRNA. The other important findings include (i) one in every twenty alternative introns is a GC-AG intron, and (ii) three of every five observed GC-AG introns are alternative isoforms.

A longitudinal analysis of cytotoxic T lymphocyte precursor frequencies to the hepatitis B virus in chronically infected patients

Individuals with acute hepatitis B virus (HBV) infection characteristically mount a strong, multispecific cytotoxic T lymphocyte (CTL) response that is effective in eradicating virus. In contrast, this response in chronic carriers is usually weak or undetectable. Since it is generally acknowledged that HBV pathogenesis is immune-mediated, the occurrence of episodes of active liver disease in many carriers suggests that these individuals can mount active CTL responses to HBV. To see whether the detection of circulating CTLs is related to these flare episodes, we have determined the CTL precursor (CTLp) frequencies to HLA-A2-restricted viral peptides in seven patients over a 12-24-month period of their disease. Limiting dilution analyses (LDA) were performed longitudinally to five epitopes comprising the viral capsid (HBc), envelope (HBs) and polymerase (pol) proteins. Assays were performed against a mixture of peptides, or against each individual peptide, to measure overall CTL activity and the multispecificity of the responses, respectively. Since two of the patients were treated with recombinant human interleukin-12 (rHuIL-12) at the time, with one individual achieving complete disease remission a year later after being treated with interferon-alpha, we were also able to examine the effects of these cytokines on HBV cytotoxicity. Our results indicate that weak but detectable CTL responses do occur in chronic carriers which are generally associated with disease flares, although CTLps were also seen occasionally during minimal disease activity. The range of specificities varied between individuals and within each individual during the course of the disease. Finally, we also provide evidence that CTL reactivity is stimulated following treatment with certain cytokines, but is dependent on the time of administration.

Derivation of the probability distribution function for the local density of states of a disordered quantum wire via the replica trick and supersymmetry

We consider the statistical properties of the local density of states of a one-dimensional Dirac equation in the presence of various types of disorder with Gaussian white-noise distribution. It is shown how either the replica trick or supersymmetry can be used to calculate exactly all the moments of the local density of states.' Careful attention is paid to how the results change if the local density of states is averaged over atomic length scales. For both the replica trick and supersymmetry the problem is reduced to finding the ground state of a zero-dimensional Hamiltonian which is written solely in terms of a pair of coupled spins which are elements of u(1, 1). This ground state is explicitly found for the particular case of the Dirac equation corresponding to an infinite metallic quantum wire with a single conduction channel. The calculated moments of the local density of states agree with those found previously by Al'tshuler and Prigodin [Sov. Phys. JETP 68 (1989) 198] using a technique based on recursion relations for Feynman diagrams. (C) 2001 Elsevier Science B.V. All rights reserved.

Generalized quasi mode theory of macroscopic canonical quantization in cavity quantum electrodynamics and quantum optics I. Theory

The quasi mode theory of macroscopic quantization in quantum optics and cavity QED developed by Dalton, Barnett and Knight is generalized. This generalization allows for cases in which two or more quasi permittivities, along with their associated mode functions, are needed to describe the classical optics device. It brings problems such as reflection and refraction at a dielectric boundary, the linear coupler, and the coupling of two optical cavities within the scope of the theory. For the most part, the results that are obtained here are simple generalizations of those obtained in previous work. However the coupling constants, which are of great importance in applications of the theory, are shown to contain significant additional terms which cannot be 'guessed' from the simpler forms. The expressions for the coupling constants suggest that the critical factor in determining the strength of coupling between a pair of quasi modes is their degree of spatial overlap. In an accompanying paper a fully quantum theoretic derivation of the laws of reflection and refraction at a boundary is given as an illustration of the generalized theory. The quasi mode picture of this process involves the annihilation of a photon travelling in the incident region quasi mode, and the subsequent creation of a photon in either the incident region or transmitted region quasi modes.

A methodology for analysis of sugarcane productivity trends - I. Analysis across districts

Historically, few articles have addressed the use of district level mill production data for analysing the effect of varietal change on sugarcane productivity trends. This appears to be due to lack of compiled district data sets and appropriate methods by which to analyse these data. Recently, varietal data on tonnes of sugarcane per hectare (TCH), sugar content (CCS), and their product, tonnes of sugar content per hectare (TSH) on a district basis, have been compiled. This study was conducted to develop a methodology for regular analysis of such data from mill districts to assess productivity trends over time, accounting for variety and variety x environment interaction effects for 3 mill districts (Mulgrave, Babinda, and Tully) from 1958 to 1995. Restricted maximum likelihood methodology was used to analyse the district level data and best linear unbiased predictors for random effects, and best linear unbiased estimates for fixed effects were computed in a mixed model analysis. In the combined analysis over districts, Q124 was the top ranking variety for TCH, and Q120 was top ranking for both CCS and TSH. Overall production for TCH increased over the 38-year period investigated. Some of this increase can be attributed to varietal improvement, although the predictors for TCH have shown little progress since the introduction of Q99 in 1976. Although smaller gains have been made in varietal improvement for CCS, overall production for CCS decreased over the 38 years due to non-varietal factors. Varietal improvement in TSH appears to have peaked in the mid-1980s. Overall production for TSH remained stable over time due to the varietal increase in TCH and the non-varietal decrease in CCS.

Radioresistant Burkitt's lymphoma cells exhibit defective Mapk signalling

Using a pair of isogenic Burkitt's lymphoma cell lines, one of which is sensitive (BL30A) and the other resistant (BL30K) to apoptosis induced by ionising radiation and exogenous ceramide, we investigated mitogen-activated protein kinase (MAPK) signalling to determine which members of this kinase family are involved in the apoptotic process in these cells. We have previously shown that BL30A cells produce ceramide after irradiation and that this does not occur in BL30K cells (Michael et at. [1997] Cancer Res 57:3600-3605). We show that p38 MAPK is activated transiently in both cells after ionising radiation. On the of her hand, although JNK is rapidly activated in both cells, this activation is only transient in the resistant cells, whereas in the sensitive cells the activation is sustained. Addition of exogenous ceramide resulted in only a transient activation of INK in both cells. Interestingly, ERK activity was decreased in BL30A cells after ceramide treatment, whereas no such decrease occurred in the resistant cells. Treatment of BL30A cells with phorbol ester before irradiation, which blocks the increase in ceramide and apoptosis, also prevents the sustained increase in JNK activity. At the same time, ERK activity is increased. Our results suggest that p38 MAPK is not required for apoptosis signalling in response to ionising radiation in Burkitt's lymphoma cells and that sustained activation of JNK is necessary for apoptosis in these cells. These results also support the hypothesis that a balance between JNK and ERK activity determines cell fate after exposure to ceramide or ionising radiation. In addition, our results suggest different signalling pathways from exogenous ceramide and radiation, supporting the concept of different intracellular pools of active ceramide. Drug Dev. Res. 52:534-541, 2001. (C) 2001 Wiley-Liss, Inc.

Using clusters of computers for large QU-GENE simulation experiments

The QU-GENE Computing Cluster (QCC) is a hardware and software solution to the automation and speedup of large QU-GENE (QUantitative GENEtics) simulation experiments that are designed to examine the properties of genetic models, particularly those that involve factorial combinations of treatment levels. QCC automates the management of the distribution of components of the simulation experiments among the networked single-processor computers to achieve the speedup.

The sequence of a 200 kb portion of a Plasmodium vivax chromosome reveals a high degree of conservation with Plasmodium falciparum chromosome 3

Within a 199 866 base pair (bp) portion of a Plasmodium vivax chromosome we identified a conserved linkage group consisting of at least 41 genes homologous to Plasmodium falciparum genes located on chromosome 3. There were no P. vivax homologues of the P. falciparum cytoadherence-linked asexual genes clag 3.2, clag 3.1 and a var C pseudogene found on the P. vivax chromosome. Within the conserved linkage group, the gene order and structure are identical to those of P. falciparum chromosome 3. This conserved linkage group may extend to as many as 190 genes. The subtelomeric regions are different in size and the P. vivax segment contains genes for which no P. falciparum homologues have been identified to date. The size difference of at least 900 kb between the homologous P. vivax chromosome and P. falciparum chromosome 3 is presumably due to a translocation. There is substantial sequence divergence with a much higher guanine + cytosine (G + C) content in the DNA and a preference for amino acids using GC-rich codons in the deduced proteins of P. vivax. This structural conservation of homologous genes and their products combined with sequence divergence at the nucleotide level makes the P. vivax genome a powerful tool for comparative analyses of Plasmodium genomes. (C) 2001 Elsevier Science B.V. All rights reserved.

Fluorescence spectrum of a two-level atom driven by a multiple modulated field

We investigate the fluorescence spectrum of a two-level atom driven by a multiple amplitude-modulated field. The driving held is modeled as a polychromatic field composed of a strong central (resonant) component and a large number of symmetrically detuned sideband fields displaced from the central component by integer multiples of a constant detuning. Spectra obtained here differ qualitatively from those observed for a single pair of modulating fields [B. Blind, P.R. Fontana, and P. Thomann, J. Phys. B 13, 2717 (1980)]. In the case of a small number of the modulating fields, a multipeaked spectrum is obtained with the spectral features located at fixed frequencies that are independent of the number of modulating fields and their Rabi frequencies. As the number of the modulating fields increases, the spectrum ultimately evolves to the well-known Mellow triplet with the sidebands shifted from the central component by an effective Rabi frequency whose magnitude depends on the initial relative phases of the components of the driving held. For equal relative phases, the effective Rabi frequency of the driving field can be reduced to zero resulting in the disappearance of fluorescence spectrum, i.e., the atom can stop interacting with the field. When the central component and the modulating fields are 180 degrees out of phase, the spectrum retains its triplet structure with the sidebands located at frequencies equal to the sum of the Rabi frequencies of the component of the driving field. Moreover, we shaw that the frequency of spontaneous emission can be controlled and switched from one frequency to another when the Rabi frequency or initial phase of the modulating fields are varied.

Measurement of qubits

We describe in detail the theory underpinning the measurement of density matrices of a pair of quantum two-level systems (qubits). Our particular emphasis is on qubits realized by the two polarization degrees of freedom of a pair of entangled photons generated in a down-conversion experiment; however, the discussion applies in general, regardless of the actual physical realization. Two techniques are discussed, namely, a tomographic reconstruction (in which the density matrix is linearly related to a set of measured quantities) and a maximum likelihood technique which requires numerical optimization (but has the advantage of producing density matrices that are always non-negative definite). In addition, a detailed error analysis is presented, allowing errors in quantities derived from the density matrix, such as the entropy or entanglement of formation, to be estimated. Examples based on down-conversion experiments are used to illustrate our results.

A zebrafish homologue of deleted in colorectal cancer (zdcc) is expressed in the first neuronal clusters of the developing brain

DCC (deleted in colon cancer), Neogenin and UNC-5 are all members of the immunoglobulin superfamily of transmembrane receptors which are believed to play a role in axon guidance by binding to their ligands, the Netrin/UNC-40 family of secreted molecules (Cell. Mol. Life Sci. 56 (1999) 62; Curr. Opin. Genet. Dev. 7 (1997) 87). Although zebrafish homologues of the Netrin family of secreted molecules have been reported, to date there has been no published description of zebrafish DCC homologues (Mol. Cell. Neurosci. 9 (1997) 293., Mol. Cell. Neurosci. I I ( 1998) 194; Mech. Dev. 62 (1997) 147). We report here the expression pattern of a zebrafish dcc (zdcc) homologue during the initial period of neurogenesis and axon tract formation within the developing central nervous system. Between 12 and 33 h post-fertilisation zdcc is expressed in a dynamic spatiotemporal pattern in all major subdivisions of the central nervous system. Double-labelling for zdcc and the post-mitotic neuronal marker HNK-1 revealed that subpopulations of neurons within the first nuclei of the zebrafish brain express zdcc. These results support our previous observation that patterning of neuronal clusters in the zebrafish brain occurs early in development (Dev. Bioi, 229 (2001) 271). (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.

Essential role of cyclization sequences in Flavivirus RNA replication

A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5'- and 3'-terminal regions of Flavivirus RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5' genomic region (first similar to 160 nucleotides) and the 3' untranslated region (last similar to 115 nucleotides) for a range of mosquito borne Flavivirus species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5' or 3') in the replicon RNA of the mosquito-borne Kunjin virus,were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5' or only the 3' cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5'- and 3'-terminal regions of three tick borne virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito borne species. They base-paired with DeltaG values of approximately -20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5' and 3' complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne Flavivirus species.

Recurrent gains and losses of large (84-109 bp) repeats in the rDNA internal transcribed spacer 2 (ITS2) of rhipicephaline ticks

We studied the internal transcribed spacer 2 (ITS2) in twenty-two spp. of ticks from the subfamily Rhipicephalinae. A 104-109 base pair (bp) region was Imperfectly repeated In most ticks studied. Mapping the number of repeat copies on to a phylogeny from the ITS2 showed that there have been many Independent gains and losses of repeats. Comparison of the sequences of the repeat copies Indicated that in most taxa concerted evolution had played little if any role in the evolution of these regions, as the copies clustered by sequence position rather than species, In our putative secondary structure, each repeat copy can fold into a distinct and almost identical stem-loop complex; a gain or loss of a repeat copy apparently does not impair the function of the ITS2 in these ticks.