Comprehensive identification and structural characterization of target components from Gelsemium elegans by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry based on accurate mass databases combined with MS/MS spectra.

This study reports an applicable analytical strategy of comprehensive identification and structure characterization of target components from Gelsemium elegans by using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QqTOF MS) based on the use of accurate mass databases combined with MS/MS spectra. The databases created included accurate masses and elemental compositions of 204 components from Gelsemium and their structural data. The accurate MS and MS/MS spectra were acquired through data-dependent auto MS/MS mode followed by an extraction of the potential compounds from the LC-QqTOF MS raw data of the sample. The same was matched using the databases to search for targeted components in the sample. The structures for detected components were tentatively characterized by manually interpreting the accurate MS/MS spectra for the first time. A total of 57 components have been successfully detected and structurally characterized from the crude extracts of G. elegans, but has failed to differentiate some isomers. This analytical strategy is generic and efficient, avoids isolation and purification procedures, enables a comprehensive structure characterization of target components of Gelsemium and would be widely applicable for complicated mixtures that are derived from Gelsemium preparations. Copyright © 2017 John Wiley & Sons, Ltd.

High performance mass spectrometry based proteomics reveals enzyme and signaling pathway regulation in neutrophils during the early stage of surgical trauma.

PURPOSE: In clinical conditions trauma is associated with high mortality and morbidity. Neutrophils play a key role in the development of multiple organ failure after trauma EXPERIMENTAL DESIGN: To have a detailed understanding of the neutrophil activation at primary stages after trauma, neutrophils are isolated from control and surgical trauma rats in this study. Extracted proteins are analyzed using nano liquid chromatography coupled with tandem mass spectrometry. RESULTS: A total of 2924 rat neutrophil proteins are identified in our analysis, of which 393 are found differentially regulated between control and trauma groups. By using functional pathways analysis of the 190 proteins up-regulated in surgical trauma, we found proteins related to transcription initiation and protein biosynthesis. On the other hand, among the 203 proteins down-regulated in surgical trauma we found enrichment for proteins of the immune response, proteasome degradation and actin cytoskeleton. Overall, enzyme prediction analysis revealed that regulated enzymes are directly involved in neutrophil apoptosis, directional migration and chemotaxis. Our observations are then confirmed by in silico protein-protein interaction analysis. CONCLUSIONS AND CLINICAL RELEVANCE: Collectively, our results reveal that neutrophils drastically regulate their biochemical pathways after the early stages of surgical trauma, showing lower activity. This implies higher susceptibility of the trauma patients to infection and bystander tissues damage.

Immunofluorescence localization of levopimaradiene/abietadiene synthase in methyl jasmonate treated stems of Sitka spruce (Picea sitchensis) shows activation of diterpenoid biosynthesis in cortical and developing traumatic resin ducts.

Conifers produce terpenoid-rich oleoresin in specialized resin ducts as a main line of defence against pests and pathogens. In spruce species (Picea spp.), axial resin ducts are either present constitutively in the cortex tissue (cortical resin ducts, CRDs) or are formed de novo as traumatic resin ducts (TRDs) in the cambial zone upon attack by insects, fungi or treatment with methyl jasmonate (MeJA). Using immunofluorescence localization we tested if previously formed CRDs respond to MeJA treatment with increased capacity for diterpenoid biosynthesis. We also tested the dynamics of diterpene synthase localization in the cambial zone. Immunofluorescence localization was performed using an antibody against a diterpene synthase, levopimaradiene/abietadiene synthase (LAS), in stem cross-sections of untreated and 0.1% MeJA-treated 4-year old Sitka spruce (P. sitchensis) trees. No fluorescence signal was observed in untreated stem cross-sections; however, signal was present 2 days after treatment with MeJA exclusively in the epithelial cells of CRDs. Fluorescence steadily increased in the CRD epithelial cells 4 and 8 days after treatment. At 8days, additional fluorescence was observed in developing epithelial cells of traumatic resin ducts TRDs in the cambial zone. These results confirm that resin duct epithelial cells are the main site of diterpene biosynthesis in Sitka spruce, diterpenoid biosynthesis is induced in CRD epithelial cells early upon treatment with MeJA, and immature developing TRD epithelial cells produce diterpene synthase enzyme. Overall, the results of this work improve our understanding of spatial and temporal patterns of induced diterpene resin acid biosynthesis in conifers.

Ruminal biohydrogenation as affected by tannins in vitro.

The aim of the present work was to study the effects of tannins from carob (CT; Ceratonia siliqua), acacia leaves (AT; Acacia cyanophylla) and quebracho (QT; Schinopsis lorentzii) on ruminal biohydrogenation in vitro. The tannins extracted from CT, AT and QT were incubated for 12 h in glass syringes in cow buffered ruminal fluid (BRF) with hay or hay plus concentrate as a substrate. Within each feed, three concentrations of tannins were used (0.0, 0.6 and 1.0 mg/ml BRF). The branched-chain volatile fatty acids, the branched-chain fatty acids and the microbial protein concentration were reduced (P < 0.05) by tannins. In the tannin-containing fermenters, vaccenic acid was accumulated (+23 %, P < 0.01) while stearic acid was reduced ( - 16 %, P < 0.0005). The concentration of total conjugated linoleic acid (CLA) isomers in the BRF was not affected by tannins. The assay on linoleic acid isomerase (LA-I) showed that the enzyme activity (nmol CLA produced/min per mg protein) was unaffected by the inclusion of tannins in the fermenters. However, the CLA produced by LA-I (nmol/ml per min) was lower in the presence of tannins. These results suggest that tannins reduce ruminal biohydrogenation through the inhibition of the activity of ruminal micro-organisms.

Prostanoids actions in cardiovascular physiopathology

Prostaglandins (PGs) and thromboxanes (TXs) play a pivotal role in cardiovascular physiopathology. They are synthesized from arachidonic acid by the enzymatic action of cyclooxygenases (COXs), leading to the production of an unstable intermediate, PGH2 that is subsequently converted to the different prostaglandins and thromboxanes (PGE2, PGD2, PGI2, PGF2α and TXA2) by the action of different synthases and isomerases. There are two well characterized COX enzymes, termed COX-1 and COX-2, with different properties. While COX-1 is expressed constitutively in most tissues and is thought to be involved in homeostatic prostanoid biosynthesis, COX-2 is transcriptionally up-regulated in response to mitogens and pro-inflammatory stimuli being the predominant isoform involved in the inflammatory response. In the cardiovascular system, prostanoids have been shown to modulate the pathogenesis of vascular diseases as thrombosis and atherosclerosis through a variety of processes, including platelet aggregation, vasorelaxation and vasoconstriction, local inflammatory response and leukocyte-endothelial cell adhesion. Multiple studies using pharmacological inhibitors and genetically deficient mice have demonstrated the importance of prostanoid-mediated actions on cardiovascular physiology. However, recent withdrawal of COX-2 selective inhibitors from the clinic because of their adverse effects in patients with potential cardiovascular risk has opened a debate about the role of COX–derived prostanoids in vascular pathologies and the benefits and risks for the use of COX inhibitors in cardiovascular diseases (AU)

Las prostaglandinas (PGs) y los tromboxanos (TXs) juegan un papel esencial en la fisiopatología cardiovascular. Estos prostanoides son sintetizados a través de la acción enzimática de las ciclooxigenasas (COXs) sobre el ácido araquidónico, lo que lleva a la producción de un intermediario inestable, la PGH2, a partir de la cual diversas sintetasas e isomerasas generarán las diferentes prostaglandinas y tromboxanos (PGE2, PGD2, PGI2, PGF2α and TXA2). Existen dos ciclooxigenasas bien caracterizadas denominadas COX-1 y COX-2, con diferentes propiedades. COX-1 se expresa constitutivamente en la mayoría de los tejidos, estando implicada en la biosíntesis de prostanoides con funciones homeostáticas. Por otro lado, la expresión de COX-2 se induce en respuesta a mitógenos y estímulos pro-inflamatorios, constituyendo la isoforma predominantemente implicada en la respuesta inflamatoria. Los prostanoides modulan la patogénesis de enfermedades vasculares como la trombosis y la aterosclerosis a través de una serie de procesos como: la agregaciónplaquetaria, la vasodilatación y vasoconstricción, y la respuesta inflamatoria local. Múltiples estudios han demostrado la importancia de las acciones mediadas por los prostanoides en la fisiopatología cardiovascular, bien mediante el uso de inhibidores farmacológicos o a través del análisis de ratones genéticamente deficientes. Sin embargo, la reciente retirada del mercado de inhibidores selectivos de COX-2 a causa de sus efectos adversos en pacientes con riesgo cardiovascular, ha abierto el debate sobre el papel de los prostanoides en la patología vascular y sobre las ventajas o inconvenientes del uso de inhibidores de COXs en las enfermedades cardiovasculares (AU)

Compositions and biological activities of essential oils of Kadsura longepedunculata and Schisandra sphenanthera.

The chemical compositions, antimicrobial activities, antioxidant activities and cytotoxicities of the essential oils isolated from the root of Kadsura longepedunculata Finet et Gagnep (KLREO) and the fruit of Schisandra sphenanthera Rehd. et Wills. (SSFEO) were investigated.The analyses of gas chromatography-mass spectrometry (GC-MS) showed that cadinane type compounds and their derivatives were rich in both oils (54.2% and 39.7%, respectively) and delta-cadinene was the major component of both oils (13.8% and 25.6%, respectively). The antimicrobial activities of both oils were evaluated against five microorganisms with the disc diffusion and the broth micro-dilution method. Results showed that Gram-positive bacteria were more sensitive to both oils than Gram-negative bacteria and the yeast. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the oil of KLREO were lower than those of SSFEO, indicating that the former possessed slightly stronger antibacterial capability than the latter. The reducing power and lipid peroxidation assays were employed to study the potential antioxidant activities of both oils. Both oils remarkably decreased the content of malondialdehyde (MDA) in rat liver homogenate in a dose dependent manner. The antioxidant activities of KLREO appeared to be more potent than that of SSFEO. The oils of KLREO and SSFEO exhibited concentration-dependent cytotoxicities and were proved to be toxic to HepG2 cells with IC(50) of 147 and 189 mug/ml, respectively.

Different susceptibility to peroxisome proliferator-induced hepatocarcinogenesis in rats with polymorphic glutathione transferase genes.

Although peroxisomal bifunctional enzyme (enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase; BE) is a positive marker for peroxisome proliferation, it is completely absent or expressed very weakly in rat hepatic preneoplastic and neoplastic lesions induced by peroxisome proliferators (PP). After administration of PP for 8-15 weeks, some rats exhibit BE-negative preneoplastic foci but other rats do not. In the present study, to investigate the involvement of glutathione S-transferase (GST) M1 gene polymorphism in interindividual differences in susceptibility to PP, we developed a method to determine the genotypes of rats. We then examined whether rats with one type encoding 198Asn-199Cys (NC-type) or another encoding 198Lys-199Ser (KS-type) exhibit differences in clofibrate (CF) susceptibility. After administration of 0.3% CF for 6 weeks or more, BE-negative foci were found immunohistochemically in KS/KS-type rats, but not in NC/NC-type rats. The number of BE-negative foci in KS/KS rats was 15.3 +/- 9.0 foci/cm2 of liver section after 6 weeks of CF administration, and the values did not alter thereafter. The mean areas of BE-negative foci in KS/KS rat livers increased during the period from 6 to 60 weeks. At weeks 30 and 60, almost all BE-negative foci exhibited a clear cell phenotype, a type of preneoplastic hepatic lesion. BE-negative foci were devoid of peroxisome proliferator-activated receptor alpha, whereas surrounding tissues were positive for the receptor. These results indicate that rats that are polymorphic for the GST M1 gene exhibit different susceptibilities to CF in vivo.

Disruption of endothelial-cell caveolin-1alpha/raft scaffolding during development of monocrotaline-induced pulmonary hypertension.

BACKGROUND: In the monocrotaline (MCT)-treated rat, there is marked stimulation of DNA synthesis and megalocytosis of pulmonary arterial endothelial cells (PAECs) within 3 to 4 days, followed by pulmonary hypertension (PH) 10 to 14 days later. Growing evidence implicates caveolin-1 (cav-1) in plasma membrane rafts as a negative regulator of promitogenic signaling. We have investigated the integrity and function of endothelial cell-selective cav-1alpha/raft signaling in MCT-induced PH. METHODS AND RESULTS: Although PH and right ventricular hypertrophy developed by 2 weeks after MCT, a reduction in cav-1alpha levels in the lung was apparent within 48 hours, declining to approximately 30% by 2 weeks, accompanied by an increase in activation of the promitogenic transcription factor STAT3 (PY-STAT3). Immunofluorescence studies showed a selective loss of cav-1alpha and platelet endothelial cell adhesion molecule-1 in the PAEC layer within 48 hours after MCT but an increase in PY-STAT3. PAECs with cav-1alpha loss displayed high PY-STAT3 and nuclear immunostaining for proliferating cell nuclear antigen (PCNA). Biochemical studies showed a loss of cav-1alpha from the detergent-resistant lipid raft fraction concomitant with hyperactivation of STAT3. Moreover, cultured PAECs treated with MCT-pyrrole for 48 hours developed megalocytosis associated with hypo-oligomerization and reduction of cav-1alpha, hyperactivation of STAT3 and ERK1/2 signaling, and stimulation of DNA synthesis. CONCLUSIONS: MCT-induced disruption of cav-1alpha chaperone and scaffolding function in PAECs likely accounts for diverse alterations in endothelial cell signaling in this model of PH.

Immunoprecipitation of DNA-protein complexes cross-linked by cis-diamminedichloroplatinum.

For the study of in vitro and in vivo DNA-protein interactions, cross-linking reactions driven by UV or formaldehyde have been frequently used, followed by standard protocols of immunoprecipitation and analysis of the DNA isolated from the complexes. Here we present a basically modified method to analyze the DNA-protein cross-linked complexes obtained by an alternative cross-linking reagent. The innovations presented here include cross-linking by cis-diamminedichloroplatinum II, a fast method to isolate DNA-protein complexes using gel-filtration chromatography, and a modified procedure to obtain specific immunocomplexes that can be analyzed either for DNA or for protein content. The application of this method to two nuclear proteins from chicken liver nuclei is described.

Leaderless polypeptides efficiently extracted from whole cells by osmotic shock.

Three molecular foldases, DsbA, DsbC, and rotamase (ppiA), exhibited the unusual property of accumulating in an osmotically sensitive cellular compartment of Escherichia coli when their signal sequences were precisely removed by mutation. A mammalian protein, interleukin-1 (IL-1) receptor antagonist, behaved in a similar fashion in E. coli when its native signal sequence was deleted. These leaderless mutants (but not two control proteins overexpressed in the same system) were quantitatively extractable from whole cells by a variety of methods generally employed in the recovery of periplasmic proteins. A series of biochemical and genetic experiments showed that (i) leaderless DsbA (but not the wild type) was retained in a nonperiplasmic location; (ii) beta-galactosidase fusions to leaderless DsbA (but not to the wild type) exhibited efficient alpha complementation; (iii) none of the leaderless mutant proteins were substantially associated with cell membranes, even when they were overexpressed in cells; and (iv) leaderless DsbA was not transported to an osmotically sensitive compartment via a secA- or ftsZ-dependent mechanism. The observation that these proteins transit to some privileged cellular location by a previously undescribed mechanism(s)--absent their normal mode of (signal sequence-dependent) translocation--was unexpected. DsbA, rotamase, and IL-1, whose tertiary structures are known, appear to be structurally unrelated proteins. Despite a lack of obvious homologies, these proteins apparently have a common mechanism for intracellular localization. As this (putative) bacterial mechanism efficiently recognizes proteins of mammalian origin, it must be well conserved across evolutionary boundaries.

Endoplasmic reticulum to Golgi trafficking in multinucleated skeletal muscle fibers.

The organization of membrane trafficking between endoplasmic reticulum and Golgi within multinucleated muscle fibers was analyzed. We found that markers for the compartment involved in endoplasmic reticulum to Golgi trafficking exhibited perinuclear as well as interfibrillar localization. Furthermore, these markers showed prominent colocalization with microtubules. To analyze membrane trafficking, we followed the temperature-controlled transport of the G protein of the mutant vesicular stomatitis virus, tsO45, in isolated myofibers. Perinuclear and cross-striated staining were seen at 39 degrees C, while at 15 degrees C a diffuse staining component appeared along a subset of interfibrillar microtubules. At 20 degrees C, bright Golgi spots were seen to be associated with microtubules that appeared as circumnuclear rings and longitudinal bundles. Beneath the motor end plate, however, the organization of the Golgi elements and microtubules was found to be distinctive. Retrograde trafficking induced by brefeldin A resulted in the disappearance of the Golgi spots throughout the myofibers and the appearance of staining along microtubules. Thus, interfibrillar membranes seem to be active in protein export, and trafficking between endoplasmic reticulum and Golgi elements occurred throughout the myofibers. The results suggest that microtubules served as tracks for the two-way trafficking between the endoplasmic reticulum and the Golgi compartment.

Antibiotic resistance in Streptomyces lividans: fluorescence assay for streptogramin B lyase.

A fluorescence assay for streptogramin B lyase, an enzyme that confers resistance to streptogramin B antibiotics, has been developed. The antibiotic substrates are fluorescent and the linear peptide products formed in the lyase-catalyzed reaction are relatively nonfluorescent. The assay has potential for assessing bacterial resistance to streptogramin B antibiotics and will be utilized to direct the purification of streptogramin B lyase from bacterial extracts.

Subcellular localization and function of melanogenic enzymes in the ink gland of Sepia officinalis.

The ink gland of the cuttlefish Sepia officinalis has traditionally been regarded as a convenient model system for investigating melanogenesis. This gland has been shown to contain a variety of melanogenic enzymes including tyrosinase, a dopachrome-rearranging enzyme and peroxidase. However, whether and to what extent these enzymes co-localize in the melanogenic compartments and interact is an open question. Using polyclonal antibodies that recognize the corresponding Sepia proteins, we have been able to demonstrate that peroxidase has a different subcellular localization pattern from tyrosinase and dopachrome-rearranging enzyme. Whereas peroxidase is located in the rough endoplasmic reticulum and in the matrix of premelanosomes and melanosomes, tyrosinase and dopachrome-rearranging enzyme are present in the rough endoplasmic reticulum-Golgi transport system, at the level of trans-Golgi cisternae, trans-Golgi network and coated vesicles, and in melanosomes on pigmented granules. These results fill a longstanding gap in our knowledge of the melanin-producing system in Sepia and provide the necessary background for dissection at the molecular level of the complex interaction between melanogenic enzymes. Moreover, the peculiar and complex organization of melanin in an invertebrate such as Sepia officinalis is surprising and could provide the basis for understanding the process in more evolved systems such as that of mammals.

Di-fluoresceinthiocarbamyl-insulin: a fluorescent substrate for the assay of protein disulfide oxidoreductase activity.

We have developed a novel method for the continuous assay of protein disulfide oxidoreductase activity using as substrate bovine pancreas insulin in which both N-terminal amino groups are chemically modified with fluorescein isothiocyanate. The reduction of intercatenary disulfide bonds of di-fluoresceinthiocarbamyl-insulin with dithiothreitol initially lowers but subsequently enhances the emission intensity. In this biphasic kinetics, the rate of increase is sensitive enough for the estimation of Escherichia coli thioredoxin concentrations from 5 nM (0.06 microgram/ml) to 500 nM (6 micrograms/ml). Neither changes of pH over a range of 6.2 to 8.4 nor neutral salts (K+, Mg2+, and Ca2+) at concentrations lower than 100 mM affect this simple reaction system. Moreover, the fluorometric method is functional for measuring the reductive capacity of Brassica napus protein disulfide isomerase. Hence, a highly reproducible and accurate one-state assay for protein disulfide oxidoreductase activity not only greatly improves the sensitivity compared to the commonly used turbidimetric assay but also represents a reliable alternative to assays based on accessory enzymes or radiolabeled substrates.

Intravillous eicosanoid compartmentalization and regulation of placental blood flow.

OBJECTIVE: To determine the roles of the eicosanoids thromboxane and prostacyclin, and their compartmentalization, in the regulation of placental blood flow. METHODS: First, the sites of production of thromboxane and prostacyclin were determined within the placental villus using immunohistochemical staining for thromboxane and prostacyclin synthetase. Second, the production of both eicosanoids was studied in cultured trophoblasts and compared with that in the villous core by measuring the metabolites thromboxane B2 and 6-keto-prostaglandin F 1 alpha. Finally, eicosanoid production was assessed in intact villi after stimulation by an acute change in oxygen content, 5% to 95%. RESULTS: Immunohistochemical staining showed that thromboxane production was primarily within the trophoblasts, whereas prostacyclin production was localized to the endothelial cells within the villi. In culture, we found preferential production of prostacyclin by the villous core cells and increased production of thromboxane by trophoblasts. Perifusion of intact villi demonstrated increased production of thromboxane by trophoblasts in response to an increase in oxygen content. Prostacyclin levels were too low to be detected. CONCLUSIONS: Placental blood flow appears to be regulated by compartmentalized eicosanoids, with thromboxane affecting primarily the maternal side of the placental circulation and prostacyclin affecting primarily the fetal side.

Comparison of the melanogenesis in human black and light brown melanocytes.

We examined how and to what extent the constitution of melanin and the expression, as well as the activity, of melanosomal proteins influence the production of melanin pigment by human black and light brown melanocytes, Mel (b) cells and Mel (l) cells, respectively. Melanin pigment in Mel (b) and Mel (l) cells consisted of a mixture of eumelanin and pheomelanin, and Mel (b) cells contained a larger amount. The signal intensity ratio of eumelanin to pheomelanin was similar in both cell types, though the two cell types differed in appearance. Tyrosinase activity and the amount of tyrosinase-related protein (TRP-1) of Mel (b) cells were higher than those of Mel (l) cells. Dopachrome tautomerase (DCT) activity and the amount of 6H5MICA were reduced in Mel (b) cells in comparison with Mel (l) cells. No significant difference in DHICA-converting activity or catechol-O-methyltransferase activity was found between Mel (b) and Mel (l) cells. There was no correlation between DHICA-converting activity and amount of TRP-1. These results suggest that the difference in the pigmentation of the two human melanocyte cell lines, Mel (b) and Mel (l), is derived from differences in the activity and expression of tyrosinase, TRP-1 and DCT, which affect the content and constitution of melanin polymers.

FMLP actions and its binding sites in isolated human coronary arteries.

The chemoattractant f-Met-Leu-Phe (FMLP) can modulate human coronary arterial tone without the involvement of peripheral leukocytes. We investigated the actions of FMLP and its cellular mechanism in human coronary arteries isolated 2-3 h after death. A single dose of FMLP (0.01-10 microM) produced transient contraction (or, followed by relaxation) responses in most human coronary rings examined. These responses to FMLP were in large part mediated by the generation of cyclooxygenase products, mainly thromboxane A2 (TXA2) and prostaglandin I2 (PGI2). Radiolabeled N-formyl hexapeptide. 125I-f-Nle-Leu-Phe-Nle-Tyr-Lys bound densely to intimal and adventitial sites that accumulated macrophages (CD68-positive) with a Kd of 14-29 nM and, further, weakly to the media with a Kd of 2.4-3.6 microM. Several cell types including macrophages, endothelial cells and smooth muscle cells were positively immunostained for both TXA2 synthase and PGI2 synthase. However, there was no significant relation between the magnitude of the responses to FMLP and dense macrophage accumulation in the intimal plaques or the adventitia. A reverse transcription-polymerase chain reaction showed predominant expression of FMLP receptor homologues, FPRH1 and FPRH2 mRNA, in human coronary medial tissues relative to that in leukocytes. In conclusion. FMLP produced transient tension changes in human coronary arteries, mainly via the generation of TXA2 and PGI2. This effect of FMLP did not appear to be mediated by the activation of densely accumulated intimal and/or adventitial macrophages, but by the activation of unidentified medial tissue cells which might have functional FMLP receptor homologues.

Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma.

Recently, major advances have been made in the identification of antigens from human melanoma which are recognized by T cells. In spite of this, little is known about the optimal ways to use these antigens to treat patients with cancer. Progress in this area is likely to require accurate preclinical animal models, but the availability of such models has lagged behind developments in human tumor immunology. Whereas many of the identified human melanoma antigens are normal tissue differentiation proteins, analogous murine tumor antigens have not yet been identified. In this paper we identify a normal tissue differentiation antigen, tyrosinase-related protein 2 (TRP-2), expressed by the murine B16 melanoma which was found by screening a cDNA library from B16 with tumor-reactive cytotoxic T lymphocytes (CTL). A peptide conforming to the predicted MHC class I H2-Kb binding motif, TRP-2181-188, was identified as the major reactive epitope within TRP-2 recognized by these anti-B16 CTLs. By site-directed mutagenesis, it was shown that alteration of this epitope eliminated recognition of TRP-2. It was further demonstrated that a CTL line raised from splenocytes by repeated stimulation in vitro with this peptide could recognize B16 tumor and was therapeutic against 3-d-old established pulmonary metastases. The use of TRP-2 in a preclinical model of tumor immunotherapy may be helpful in suggesting optimal vaccination strategies for cancer therapy in patients.

Protein disulfide isomerase and newly synthesized procollagen chains form higher-order structures in the lumen of the endoplasmic reticulum.

A number of proteins that act as necessary catalysts for correct protein folding and oligomerization in the endoplasmic reticulum (ER) are known to be retained in the organelle via the KDEL-receptor mediated retrieval mechanism. However, a complementary system that may help to retain these proteins in the organelle lumen has been suggested to exist and likely involves physical protein-protein interactions at the level of endoplasmic reticulum (ER) itself. In this report, we provide both morphological and biochemical evidence in support of this proposal. We show that in collagen-secreting human skin fibroblasts, protein disulfide isomerase and newly synthesized procollagen chains exist predominantly in an "aggregated" state, and form a reticular-like matrix in the ER lumen in vivo. The size of the aggregates was found to be variable, and may exceed 1.5 million Da. Aggregate formation appeared to be transient and to involve multiple types of protein-protein interactions, including formation of aberrant disulfide bonds. Association of protein disulfide isomerase, on the other hand, was found to require at least partly function-related disulfide bonds. These results support the existence of a reticular-like matrix in the ER lumen, and suggest that aggregation may be part of the normal maturation pathway during collagen biosynthesis.

Immunofluorometric assay of prostaglandin D synthase in human tissue extracts and fluids.

A two-site sandwich-type assay for human prostaglandin D (PGD) synthase (beta-trace) was developed with two monoclonal antibodies and using time-resolved fluorometry as the detection technique. The assay is precise (CVs < 10%), accurate, and highly specific for PGD synthase and has a detection limit of 0.05 microgram/L. Using this assay, we measured PGD synthase concentrations in serum, urine, amniotic fluid, cerebrospinal fluid (CSF), seminal plasma, breast cyst fluid, breast discharge fluid, breast milk, and breast tumor extracts. The highest concentrations were found in CSF. We identified proteolytic degradation of PGD synthase in amniotic fluid. Fetal tissues contained various amounts of the enzyme, with the highest values being found in brain and heart. In placental extracts, PGD synthase content was greatest at 11-28 weeks of gestation-in accordance with the concentrations measured in amniotic fluids for this gestational period. We conclude that PGD synthase is ubiquitous and is present in many fluids and tissues of adults and fetuses. This first quantitative and sensitive assay of PGD synthase should facilitate expansion of knowledge on this enzyme and possibly will have applications for diagnosis and monitoring of human diseases.