Generation and phenotypic characterization of new human ovarian cancer cell lines with the identification of antigens potentially recognizable by HLA-restricted cytotoxic T cells

This study describes a simple method for long-term establishment of human ovarian tumor lines and prediction of T-cell epitopes that could be potentially useful in the generation of tumor-specific cytotoxic T lymphocytes (CTLs), Nine ovarian tumor lines (INT.Ov) were generated from solid primary or metastatic tumors as well as from ascitic fluid, Notably all lines expressed HLA class I, intercellular adhesion molecule-1 (ICAM-1), polymorphic epithelial mucin (PEM) and cytokeratin (CK), but not HLA class II, B7.1 (CD80) or BAGE, While of the 9 lines tested 4 (INT.Ov1, 2, 5 and 6) expressed the folate receptor (FR-alpha) and 6 (INT.Ov1, 2, 5, 6, 7 and 9) expressed the epidermal growth factor receptor (EGFR); MAGE-1 and p185(HER-2/neu) were only found in 2 lines (INT.Ov1 and 2) and GAGE-1 expression in 1 line (INT.Ov2). The identification of class I MHC ligands and T-cell epitopes within protein antigens was achieved by applying several theoretical methods including: 1) similarity or homology searches to MHCPEP; 2) BIMAS and 3) artificial neural network-based predictions of proteins MACE, GAGE, EGFR, p185(HER-2/neu) and FR-alpha expressed in INT.Ov lines, Because of the high frequency of expression of some of these proteins in ovarian cancer and the ability to determine HLA binding peptides efficiently, it is expected that after appropriate screening, a large cohort of ovarian cancer patients may become candidates to receive peptide based vaccines. (C) 1997 Wiley-Liss, Inc.

Identification of the regulatory subunit of Arabidopsis thaliana acetohydroxyacid synthase and reconstitution with its catalytic subunit

Acetohydroxyacid synthase (EC 4.1.3.18; AHAS) catalyzes the initial step in the formation of the branched-chain amino acids. The enzyme from most bacteria is composed of a catalytic subunit, and a smaller regulatory subunit that is required for full activity and for sensitivity to feedback regulation by valine. A similar arrangement was demonstrated recently for yeast AHAS, and a putative regulatory subunit of tobacco AHAS has also been reported. In this latter case, the enzyme reconstituted from its purified subunits remained insensitive to feedback inhibition, unlike the enzyme extracted from native plant sources. Here we have cloned, expressed in Escherichia coil, and purified the AHAS regulatory subunit of Ambidopsis thaliana. Combining the protein with the purified A. thaliana catalytic subunit results in an activity stimulation that is sensitive to inhibition by valine, leucine, and isoleucine. Moreover, there is a strong synergy between the effects of leucine and valine, which closely mimics the properties of the native enzyme. The regulatory subunit contains a sequence repeat of approximately 180 residues, and we suggest that one repeat binds leucine while the second binds valine or isoleucine. This proposal is supported by reconstitution studies of the individual repeats, which were also cloned, expressed, and purified. The structure and properties of the regulatory subunit are reminiscent of the regulatory domain of threonine deaminase (EC 4.2.1.16), and it is suggested that the two proteins are evolutionarily related.

Making decisions with the future in mind: Developmental and comparative identification of mental time travel

Mechanisms that produce behavior which increase future survival chances provide an adaptive advantage. The flexibility of human behavior is at least partly the result of one such mechanism, our ability to travel mentally in time and entertain potential future scenarios. We can study mental time travel in children using language. Current results suggest that key developments occur between the ages of three to five. However, linguistic performance can be misleading as language itself is developing. We therefore advocate the use of methodologies that focus on future-oriented action. Mental time travel required profound changes in humans' motivational system, so that current behavior could be directed to secure not just present, but individually anticipated future needs. Such behavior should be distinguishable from behavior based on current drives, or on other mechanisms. We propose an experimental paradigm that provides subjects with an opportunity to act now to satisfy a need not currently experienced. This approach may be used to assess mental time travel in nonhuman animals. We conclude by describing a preliminary study employing an adaptation of this paradigm for children. (c) 2005 Elsevier Inc. All rights reserved.

Butyrylcholinesterase: Association with the metabolic syndrome and identification of 2 gene loci affecting activity

Background: Plasma cholinesterase activity is known to be correlated with plasma triglycerides, HDL- and LDL-cholesterol, and other features of the metabolic syndrome. A role in triglyceride metabolism has been proposed. Genetic variants that decrease activity have been studied extensively, but the factors contributing to overall variation in the population are poorly understood. We studied plasma cholinesterase activity in a sample of 2200 adult twins to assess covariation with cardiovascular risk factors and components of the metabolic syndrome, to determine the degree of genetic effects on enzyme activity, and to search for quantitative trait loci affecting activity. Methods and Results: Cholinesterase activity was lower in women than in men before the age of 50, but increased to activity values similar to those in males after that age. There were highly significant correlations with variables associated with the metabolic syndrome: plasma triglyceride, HDL- and LDL-cholesterol, apolipoprotein B and E, urate, and insulin concentrations; gamma-glutamyltransferase and aspartate and alanine aminotransferase activities; body mass index; and blood pressure. The heritability of plasma cholinesterase activity was 65%. Linkage analysis with data from the dizygotic twin pairs showed suggestive linkage on chromosome 3 at the location of the cholinesterase WHO gene and also on chromosome 5. Conclusions: Our results confirm and extend the connection between cholinesterase, cardiovascular risk factors, and metabolic syndrome. They establish a substantial heritability for plasma cholinesterase activity that might be attributable to variation near the structural gene and at an independent locus. (c) 2006 American Association for Clinical Chemistry.

Extending the theory of Owicki and Gries with asynchronous message passing

We describe an extension of the theory of Owicki and Gries (1976) to a programming language that supports asynchronous message passing based on unconditional send actions and conditional receive actions. The focus is on exploring the fitness of the extension for distributed program derivation. A number of experiments are reported, based on a running example problem, and with the aim of exploring design heuristics and of streamlining derivations and progress arguments.

Common chromosomal fragile site FRA16D sequence: identification of the FOR gene spanning FRA16D and homozygous deletions and translocation breakpoints in cancer cells

Fluorescence in situ hybridization of a tile path of DNA subclones has previously enabled the cytogenetic definition of the minimal DNA sequence which spans the FRA16D common chromosomal fragile site, located at 16q23.2. Homozygous deletion of the FRA16D locus has been reported in adenocarcinomas of stomach, colon, lung and ovary. We have sequenced the 270 kb containing the FRA16D fragile site and the minimal homozygously deleted region in tumour cells. This sequence enabled localization of some of the tumour cell breakpoints to regions which contain AT-rich secondary structures similar to those associated with the FRA10B and FRA16B rare fragile sites. The FRA16D DNA sequence also led to the identification of an alternatively spliced gene, named FOR (fragile site FRA16D oxidoreductase), exons of which span both the fragile site and the minimal region of homozygous deletion. In addition, the complete DNA sequence of the FRA16D-containing FOR intron reveals no evidence of additional authentic transcripts. Alternatively spliced FOR transcripts (FOR I, FOR II and FOR III) encode proteins which share N-terminal WW domains and differ at their C-terminus, with FOR III having a truncated oxidoreductase domain. FRA16D-associated deletions selectively affect the FOR gene transcripts. Three out of five previously mapped translocation breakpoints in multiple myeloma are also located within the FOR gene. FOR is therefore the principle genetic target for DNA instability at 16q23.2 and perturbation of FOR function is likely to contribute to the biological consequences of DNA instability at FRA16D in cancer cells.

Identification of the glycosaminoglycan keratan sulfate in the prostatic secretory cell

BACKGROUND. Prostate secretory granules (PSG) represent the basic secretory unit of the prostate gland, containing many of its exocrine proteases. Recent analysis of intraluminal corpora amylacea, a proposed by-product of PSG secretion, detected sulfated glycosaminoglycans (GAG) possibly keratan sulfate (KS),indicating a secretory mechanism for GAG in the human prostate surface epithelial cell. METHODS. Immunostains using anti-KS and anti-prostate-specific antigen (PSA) were evaluated on 10 sequential radical prostatectomy specimens, three of which had received neoadjuvant antiandrogen therapy. Extracts of normal secretory tissue as well as a sample composed almost entirely of prostatic stroma were subjected to Western blot analysis, using the same antibody panel. RESULTS. Keratan sulfate secretion from the normal prostate epithelial cell has been confirmed and correlates, as does PSA, with the presence of cytoplasmic PSG. No such correlation exists in most adenocarcinomas or in benign epithelium after androgen ablation. Western blot analyses confirmed tissue immunostains and demonstrated a secretory proteoglycan of 70-95 kDa. CONCLUSIONS. Recognition of PSG heralds a novel secretory mechanism within the human prostate gland that is linked to the secretion of KS. The role of KS in normal prostate secretion remains unknown, although it appears downregulated in neoplastic and androgen-ablated cells. (C) 2000 Wiley-Liss, Inc.

Identification of the coupling between skeletal muscle store-operated Ca2+ entry and the inositol trisphosphate receptor

Examination of store-operated Ca2+ entry (SOC) in single, mechanically skinned skeletal muscle cells by confocal microscopy shows that the inositol 1,4,5-trisphosphate (IP3) receptor acts as a sarcoplasmic reticulum [Ca2+] sensor and mediates SOC by physical coupling without playing a key role in Ca2+ release from internal stores, as is the case with various cell types in which SOC was investigated previously. The results have broad implications for understanding the mechanism of SOC that is essential for cell function in general and muscle function in particular. Moreover, the study ascribes an important role to the IN receptors in skeletal muscle, the role of which with respect to Ca2+ homeostasis was ill defined until now.

Identification of the alpha(6) integrin as a candidate receptor for papillomaviruses

Papillomaviruses (PVs) bind in a specific and saturable fashion to a range of epithelial and other cell lines. Treatment of cells with trypsin markedly reduces their ability to bind virus particles, suggesting that binding is mediated via a cell membrane protein. We have investigated the interaction bf human PV type 6b L1 virus-like particles (VLPs) with two epithelial cell lines, CV-1 and HaCaT, which bind VLPs, and a B-cell line (DG75) previously shown not to bind VLPs. Immunoprecipitation of a mixture of PV VLPs with [S-35]methionine-labeled cell extracts and with biotin-labeled cell surface proteins identified four proteins from CV-1 and HaCaT cells of 220, 120, 87, and 35 kDa that reacted with VLPs and were not present in DG75 cells. The alpha(6) beta(4) integrin complex has subunits corresponding to the VLP precipitated proteins, and the tissue distribution of this complex suggested that it was a candidate human PV receptor. Monoclonal antibodies (MAbs) to the alpha(6) or beta(4) integrin subunits precipitated VLPs from a mixture of CV-1 cell proteins and VLPs, whereas MAbs to other integrin subunits did not. An alpha(6) integrin-specific MAb (GoH3) inhibited VLP binding to CV-1 and HaCaT cells, whereas an anti-beta(4) integrin MAb and a range of integrin-specific and other MAbs did not. Furthermore, human laminin, the natural ligand for the alpha(6) beta(4) integrin, was able to block VLP binding. By use of sections of monkey esophagus, the distribution of alpha(6), integrin expression in the basal epithelium was shown to coincide with the distribution of bound VLPs. Taken together, these data suggest that VLPs bind specifically to the alpha(6) integrin subunit and that integrin complexes containing alpha(6) integrin complexed with either beta(1) or beta(4) integrins may act as a receptor for PV binding and entry into epithelial cells.

Characterization of mouse Frizzled-3 expression in hair follicle development and identification of the human homolog in keratinocytes

Frizzled genes encode a family of Wnt ligand receptors, which have a conserved cysteine-rich Wnt binding domain and include both transmembrane and secreted forms. Work by others has shown that experimental perturbation of Wnt signaling results in aberrant hair formation, hair growth, and hair structure. To date, however, there is no information on the contribution of individual Frizzled proteins to hair development. We now report that Frizzled-3 expression in skin is restricted to the epidermis and to the developing hair follicle. Northern analysis on total mouse skin mRNA revealed a single Frizzled-3 transcript of 3.7 kb. Reverse transcription-polymerase chain reaction and in situ hybridization analysis revealed Frizzled-3 expression in epidermal and hair follicle keratinocytes. Frizzled-3 transcripts are first detected in discrete foci in the developing epidermis of 13 d embryos and later in the hair follicle placodes of 15 d embryos, suggesting a role for this Frizzled isoform in follicle development. In 17 d embryos and id old newborn mice Frizzled-3 expression is limited to suprabasal keratinocytes and is not seen in pelage follicles until 3 d postpartum. In 7 d old neonatal skin, Frizzled-3 is expressed throughout the epidermis and in the outer cell layers of hair follicles. We have also identified the mRNA encoding human Frizzled-3 in epidermal keratinocytes and in the HaCaT keratinocyte cell line. Human Frizzled-3 mRNA encodes a 666 amino acid protein with 97.8% identity to the mouse protein. The human Frizzled-3 gene was mapped using a radiation-hybrid cell line panel to the short arm of chromosome 8 between the markers WI-1172 and WI-8496 near the loci for the Hypotrichosis of Marie Unna and Hairless genes.

Isolation and identification of the toxic peptides from Lophyrotoma zonalis (Pergidae) sawfly larvae

The broad-leaved paper bark tree Melaleuca quinquenervia (Cav) (Myrtaceae) was introduced into Florida (USA) early in this century it has proliferated to such an extent that urgent measures are now required to control it. The sawfly Lophyrotoma zonalis (Pergidae) has been introduced as a possible biological control agent due to its ability to defoliate M. quinquenervia. Because toxic D-amino acid- containing peptides have been isolated from some sawfly species, L. zonalis larvae were processed using the previously reported method for the recovery of these compounds. The toxins lophyrotomin (as the free C-terminal acid) and a mixture of pergidin and Val(4)-pergidin were isolated at 0.36 and 0.43% yield of the dried larvae, respectively. Both compounds when dosed intraperitoneally to C57/B16 male mice were hepatotoxic with lowest lethal doses of 8 and 32 mg/kg, respectively. The pathology of the liver was different for each compound, with the lophyrotomin free acid causing a periportal haemorrhagic necrosis and the pergidin causing a periacinar coagulative necrosis. (C) 2001 Elsevier Science Ltd. All rights reserved.

Isolation and identification of the cyanotoxin cylindrospermopsin and deoxy-cylindrospermopsin from a Thailand strain of Cylindrospermopsis raciborskii (Cyanobacteria)

A strain of Cylindrospermopsis (Cyanobacteria) isolated from a fishpond in Thailand was examined for its taxonomy based upon morphology and 16S rRNA gene sequence. It was also examined for production of the hepatotoxic cyanotoxin called cylindrospermopsin (CYN) and deoxycylindrospermopsin (deoxy-CYN). The strain (CY-Thai) was identified as C. raciborskii (Woloszynska) Seenaya and Subba Raju based upon morphological examination which was confirmed by 16S rRNA gene sequences and phylogenetic comparisons based upon its 16S rRNA gene. The alkaloid heptatotoxin CYN was confirmed using mouse bioassay, HPLC and HPLC-MS/MS while deoxy-CYN was confirmed using HPLC-MS/MS. The mouse bioassay gave a minimum lethal dose at 250 mg dry weight cells/kg body weight within 24 h and 125 mg/kg at 72 h, with signs of poisoning the same as in literature reports for CYN. HPLC chromatographic comparison of the CY-Thai toxin with standard CYN gave the same retention time and an absorbance maximum at 262 nm. HPLC-MS/MS confirmed the presence of CYN (M + H 416) and deoxy-CYN (M + H 400). The CYN content in strain CY-Thai was estimated at 1.02 mg/g and approximately 1/10 of this amount for deoxy-CYN. This is the first report from Asia of a CYN, deoxy-CYN producing Cylindrospermopsis raciborskii. (C) 2001 Elsevier Science Ltd. All rights reserved.

The identification of the transcriptional regulator CRP in Aeromonas hydrophila JMP636 and its involvement in amylase production and the 'acidic toxicity' effect

Aims: The physiological examination of amylase production by Aeromonas hydrophila JMP636 and identification of the mechanism of regulation. Methods and Results: Aeromonas hydrophila JMP636 was grown with single, then dual carbon sources; the growth cycle was followed and amylase activity throughout was monitored. The levels of cAMP, a known secondary messenger for the regulatory gene crp, were also examined. Amylase activity was regulated by catabolite repression. Physiological studies revealed that JMP636 exhibited both diauxic growth, with two carbon sources, and the 'acid toxicity' effect on glucose. The crp gene was cloned, expressed and inactivated from the JMP636 chromosome. Catabolite repression of amylase production and the 'acid toxicity' effect both require crp and were linked to cAMP levels. Conclusions: Regulation of amylase production was predicted to follow the model CRP-mediated cAMP-dependent Escherichia coli catabolite regulation system. Significance and Impact of the Study: This work provides an understanding of the physiology of the opportunistic pathogen Aer. hydrophila through identification of the mechanism of catabolite repression of amylase production and the existence of crp within this cell. It also provides a broader knowledge of global gene regulation and suggests regulatory mechanisms of other Aer. hydrophila gene/s.

Identification of the enamelin (g.8344delG) mutation in a new kindred and presentation of a standardized ENAM nomenclature

The amelogenesis imperfectas (Al) area geneticatly heterogeneous group of diseases that result in defective development of tooth enamel. Although X-linked, autosomal. dominant and autosomal. recessive forms of Al have been clinically characterized, only two genes (AMELX and ENAM) have been associated with Al. To date, three enamelin (ENAM) mutations have been identified. These mutations cause phenotypically diverse forms of autosomal. dominant Al. Detailed phenotype-genotype correlations have not been performed for autosomal. dominant Al due to ENAM mutations. We identified a previously unreported kindred segregating for the ENAM mutation, g.8344delG. Light and electron microscopy analyses of unerupted permanent teeth show the enamel is markedly reduced in thickness, Lacks a prismatic structure and has a laminated appearance. Taken together these histological features support the enamelin protein as being critical for the development of a normal. enamel. thickness and that it Likely has a role in regulating c-axis crystallite growth. Because there is growing molecular and phenotypic diversity in the enamelin defects, it is critical to have a nomenclature and numbering system for characterizing these conditions. We present a standardized nomenclature for ENAM mutations that will allow consistent reporting and communication. (C) 2003 Elsevier Science Ltd. All rights reserved.

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

The albA gene from Klebsiella oxytoca encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30%, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors. The albA gene was subsequently cloned from K oxytoca ATCC 13182(T) to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His(78), HiS(125), HiS(141) and His(189)) in the corrected sequence for AlbA. Treatment of AlbA with diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, reduced binding activity by about 95%. DEPC treatment increased absorbance at 240-244 nm by an amount indicating conversion to N-carbethoxyhistidine of a single histidine residue per AlbA molecule. Pretreatment with albicidin protected AlbA against modification by DEPC, with a 1 : 1 molar ratio of albicidin to the protected histidine residues. Based on protein secondary structure and amino acid surface probability indices, it is predicted that HiS125 might be the residue required for albicidin binding. Mutation of HiS125 to either alanine or leucine resulted in about 32% loss of binding activity, and deletion of HiS125 totally abolished binding activity. Mutation of HiS125 to arginine and tyrosine had no effect. These results indicate that HiS125 plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.