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.

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.

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.

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.

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.

Distinct stages of melanocyte differentiation revealed by anlaysis of nonuniform pigmentation patterns.

The injection of an antagonistic anti-murine c-kit monoclonal antibody ACK2 during mouse embryonic development produced three distinctive pigmentation patterns on the coat of the offspring. Pattern 1 consisted of pigmentation in craniofacial and caudal regions and was induced by an ACK2 injection between 9.5 and 11.5 days post coitum (dpc). In pattern 2, the entire coat was unpigmented and was induced by the injection at around 13.0 dpc. Pattern 3 consisted of pigmented patches spreading ventrolaterally from the dorsoanterior trunk regions towards the anterior and posterior directions and it was induced by ACK2 administered at 14.5-15.0 dpc. We investigated the embryological basis of these nonuniform pigmentation patterns to elucidate the process of melanoblast differentiation between lineage commitment and colonization into developing hair follicles. The results showed the following. (1) Melanocyte differentiation at the embryonic stage from 10.5 to 12.5 dpc progresses in a spatially nonuniform fashion, being faster in the craniofacial and caudal regions than in the trunk; pattern 1 reflects this. (2) Melanoblasts are activated to proliferate synchronously upon entering into the epidermis; pattern 2 correlates with this process. (3) c-kit functions as a survival signal for proliferating melanoblasts in the epidermis. (4) The melanoblasts that enter developing hair follicles can survive without a c-kit signal; pattern 3 essentially represents the hair follicles colonized by these cells. Analysis of the melanoblast distribution of ls/ls embryos that bear a loss-of-function mutation in the endothelin 3 gene suggested that endothelin 3 is required for early melanoblast differentiation before entering into the epidermis, whereas proliferation in the epidermis takes place without this molecule. Based on these data, we propose 4 distinct steps of embryonic melanocyte differentiation: (1) migration in the dermis, which requires both c-kit and endothelin 3; (2) a state before epidermal entry that is resistant to anti-c-kit mAb; (3) cell proliferation after entering the epidermal layer, which requires c-kit and endothelin receptor B but not endothelin 3 and (4) integration into developing hair follicles, which renders melanoblasts resistant to anti-c-kit mAb. Thus, melanoblast differentiation proceeds by alternately repeating c-kit -dependent and c-kit-independent stages and c-kit functions as a survival factor for the proliferating melanoblasts.

Astrocytes synthesize and secrete prostaglandin D synthetase in vitro.

Prostaglandin D synthetase [PGD-S, prostaglandin-H2 D-isomerase, (5Z, 13E)-(15S)-9alpha, 11 alpha-epidioxy-15-hyrdroxyprosta-5,13-dienoate D-isomerase, EC 5,3,99,2], an enzyme that catalyzes the formation of prostaglandin D2, was originally isolated from homogenates of rat brain and spleen and is known to be a membrane-bound enzyme. Subsequent immunohistochemical studies have shown that PGD-S is associated with neurons in the brain of immature rats, whereas in adult rats it is associated with oligodendrocytes. Several recent studies have shown that the beta-trace protein isolated from human cerebrospinal fluid (CSF), the second most abundant protein in human CSF after albumin, is equivalent to PGD-S. In this paper, we report the preparation of a monospecific polyclonal antibody against purified PGD-S isolated from human CSF and the establishment of a specific radioimmunoassay for this protein. Using this radioimmunoassay in conjunction with immunoblot analysis, PGD-S was detected in various biological fluids including serum, aqueous humor, and rete testis fluid. In addition, an antibody prepared against human PGD-S partially cross-reacted with the PGD-S in the rat and ram. Using a monospecific polyclonal antibody prepared against purified rat PGD-S isolated from rat CSF in conjunction with [35S]methionine incorporation and immunoprecipitation techniques, it was shown for the first time that PGD-S is actively synthesized and secreted by astrocytes cultured in vitro, suggesting the astrocyte is the cellular origin of PGD-S in the CSF. The identification of the astrocyte as the cellular origin of this unique enzyme will allow the use of an in vitro system to study its regulation.

Localization of protein disulfide isomerase to the external surface of the platelet plasma membrane.

Protein disulfide isomerase (PDI) is an enzyme that catalyzes the formation as well as the isomerization of disulfide bonds. In this study, antibodies against PDI were used to show PDI antigen on the platelet surface by indirect immunofluorescence microscopy and by flow cytometry. The platelets were not activated, as evidenced by the absence of staining by an antibody against P-selectin. Permeabilized platelets showed little cytosolic PDI by indirect immunofluorescence microscopy, suggesting that the majority of platelet PDI is localized to the platelet surface. PDI activity against "scrambled" RNase was shown with intact platelets. The activity was inhibited by inhibitors of PDI and by an antibody against PDI. Other blood cells showed little PDI. Platelet surface PDI may play a role in the various physiological and pathophysiologic processes in which platelets are involved.

Secretion, surface localization, turnover, and steady state expression of protein disulfide isomerase in rat hepatocytes.

Protein disulfide isomerase in isolated rat hepatocytes was present at a concentration of 7 micrograms/mg cell protein, representing a approximately 2-fold enrichment compared to isolated hepatic non-parenchymal cells. Though localized mainly in microsomal fractions of hepatocytes, direct immunofluorescence and cell surface radioiodination followed by immunoprecipitation revealed the presence of M(r) 57,000 disulfide isomerase at the cell surface. Electrostatic interaction of the protein with the cell surface was suggested by susceptibility to carbonate washing. Metabolic radiolabeling and immunoprecipitation studies also indicated that some of the newly synthesized M(r) 57,000 disulfide isomerase was secreted. Treatment of cells with colchicine markedly reduced the recovery of disulfide isomerase from the media, indicating microtubular-directed secretion of the protein. Partial staphlococcal V8 proteolytic digestion of the secreted protein revealed a peptide pattern similar to that of the cellular protein. Immunoprecipitation with antibody specific to the -KDEL peptide retention sequence confirmed the presence of this sequence in the secreted protein. Studies of the turnover of disulfide isomerase revealed a half-life of approximately 96 h. Treatment of cells with tunicamycin or heat shock resulted in an increased recovery of newly synthesized disulfide isomerase from cell lysates but diminished recovery from the media. The secretion and cell surface distribution of disulfide isomerase in hepatocytes may be important for the pathogenesis of immune mediated liver injury.

Prostacyclin (PGI2) synthase is a constitutively expressed enzyme in human endothelial cells.

Biogenesis of prostanoids is under the control of some polypeptide growth factors. Cytosolic phospholipase A2, a form specific for arachidonic acid containing phospholipids, is activated by a translocation mechanism regulated by growth factors, while prostaglandin H synthase isoforms are induced de novo in several cell types. No information is available as far as PGI2 synthase is concerned. Human umbilical vein endothelial cells were cultured under conditions favoring proliferation or differentiation or capillary-like network formation in the presence of collagen gels. Basic fibroblast growth factor (bFGF 0.5-4 ng/ml) was used as a mitogen, interleukin-1 alpha (IL-1 alpha 10-60 UI/ml) as a differentiating agent, and prostacyclin (PGI2) biosynthesis was evaluated. Under the first condition, basal PGI2 production was unaffected while, in the presence of IL-1 alpha, a marked stimulation of PGI2 synthesis was observed. It is known that IL-1 alpha is a potent inducer of PGH synthase, while it is not known whether PGI2 synthase is also induced. Two lines of evidence indicate that PGI2 synthase is a constitutively expressed not inducible enzyme: (a) proliferating nonproducing cells when added with PGH2 produce an amount of PGI2 not different from the amount produced by cells stimulated with IL-1 alpha; (b) under this condition PGI2 synthase was immunodetectable either by immunofluorescence detected by confocal microscopy or by ELISA and, on microsomes isolated from endothelial cells, by Western blotting. It is concluded that the limiting step in the conversion arachidonate-PGI2 is represented solely by the level of PGH synthase. These results strongly suggest, but do not prove, the constitutive nature of the enzyme. The final demonstration requires the availability of a probe to detect mRNA level, a trial we are carrying out at the moment.

Complex formation between mushroom tyrosinase and Manduca dopachrome isomerase.

Melanin biosynthesis in animals is initiated by the ubiquitously present tyrosinase and is aided by dopachrome isomerase. We have characterized a novel dopachrome isomerase (decarboxylating) from the hemolymph of Manduca sexta that generates a new quinone methide intermediate during melanogenesis (Sugumaran, M. and Semensi, V. (1991) J. Biol. Chem. 266, 6073-6078). This enzyme has the ability to form a complex with mushroom tyrosinase as judged by a number of physicochemical studies. The isomerase exhibited a marked inhibitory effect on tyrosinase and tyrosinase reciprocated by inhibiting the isomerase. While the isomerase showed no activity toward preformed dopaminechrome, it readily influenced the stability of dopaminechrome generated in situ by tyrosinase. Moreover, mushroom tyrosinase, which lacked specific binding to Concanavalin A Sepharose column, after complexing with the isomerase exhibited binding to this column. The complex formation also affected the pI value as well as mobility on a size exclusion column of these enzymes. Enzymes executing sequential metabolic transformation are known to form complexes called metabolons. Based on these above studies, it is concluded that both the enzymes involved in insect melanogenic pathway--phenoloxidase and dopachrome isomerase--are able to form a metabolon complex.

Cultured lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis have a diminished capacity to synthesize prostaglandin E2 and to express cyclooxygenase-2.

Prostaglandin E2 (PGE2) inhibits fibroblast proliferation and collagen synthesis. In this study, we compared lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis (F-IPF) and from patients undergoing resectional surgery for lung cancer (F-nl) with respect to their capacity for PGE2 synthesis and their expression and regulation of cyclooxygenase (COX) proteins. Basal COX activity, assessed by quantitating immunoreactive PGE2 synthesized from arachidonic acid, was twofold less (P < 0.05) in F-IPF than F-nl. In F-nl, incubation with the agonists PMA, LPS, or IL-1 increased COX activity and protein expression of the inducible form of COX, COX-2, and these responses were inhibited by coincubation with dexamethasone. By contrast, F-IPF failed to demonstrate increases in COX-2 protein expression or COX activity in response to these agonists. Under conditions of maximal induction, COX activity in F-IPF was sixfold less than that in F-nl (P < 0.05). Our data indicate that F-IPF have a striking defect in their capacity to synthesize the antiinflammatory and antifibrogenic molecule PGE2, apparently because of a diminished induction of COX-2 protein. This reduction in the endogenous capacity of F-IPF to down-regulate their function via PGE2 may contribute to the inflammatory and fibrogenic response in IPF. Moreover, we believe that this represents the first description of a defect in COX-2 expression in association with a human disease.

Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A.

Poliovirus RNA replication occurs on the surface of membranous vesicles that proliferate throughout the cytoplasm of the infected cell. Since at least some of these vesicles are thought to originate within the secretory pathway of the host cell, we examined the effect of poliovirus infection on protein transport through the secretory pathway. We found that transport of both plasma membrane and secretory proteins was inhibited by poliovirus infection early in the infectious cycle. Transport inhibition did not require viral RNA replication or the inhibition of host cell translation by poliovirus. The viral proteins 2B and 3A were each sufficient to inhibit transport in the absence of viral infection. The intracellular localization of a secreted protein in the presence of 3A with the endoplasmic reticulum suggested that 3A directly blocks transport from the endoplasmic reticulum to the Golgi apparatus.

High-performance liquid-chromatographic analysis of dopachrome and dihydroxyphenylalanine.

A high-performance liquid-chromatographic system for the determination of dopachrome and dihydroxy-phenylalanine (dopa) is described. The retention of dopa and dopachrome on C18 reversed-phase columns was investigated as a function of pH in the mobile phase, and as expected the capacity factors were found to be pH dependent. The chromatographic behavior is explained by the change in net charge and polarity of dopachrome and dopa when pH varies. Satisfactory separation of dopachrome and dopa was obtained. An advantage of the method is that the measurements of dopachrome stability and disappearance are uninfluenced by concomitant formation of melanochromes which, however, is the case when the disappearance is followed by measurement of the decrease in absorbance at 475 nm. The utility of the method is illustrated by following the disappearance of dopachrome as a measure of dopachrome tautomerase activity.

Lysyl hydroxylase, a collagen processing enzyme, exemplifies a novel class of luminally-oriented peripheral membrane proteins in the endoplasmic reticulum.

Lysyl hydroxylase (LH), an enzyme required early during collagen biosynthesis, appears to be exceptional among proteins that are thought to be residents of the endoplasmic reticulum (ER). It is a homodimer and does not contain either of the two previously characterized ER-specific retention motifs (KDEL or the double lysine motif) in its primary structure. We now show that LH, nevertheless, resides in the lumen of the ER. In immunofluorescence experiments, LH co-localizes with a KDEL-containing protein, protein disulfide isomerase (PDI), and also co-sediments with it after fractionation of subcellular organelles by sucrose density gradient centrifugation. In addition, LH seems to be stress-inducible. In one respect, however, LH differs from PDI and other known luminal proteins in the organelle. It is found in situ only in association with the ER membranes. Our cell fractionation and Triton X-114 phase separation experiments suggest that it binds to the membranes via weak electrostatic interactions. LH can thus be regarded as a first luminally-oriented "peripheral membrane" protein which has been characterized in the ER. The results suggest a novel possibility by which ER lumen can acquire its specific protein components from the bulk flow.

Expression of peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase enzyme and its mRNA in peroxisome proliferator-induced liver tumors.

We have examined ciprofibrate and dehydroepiandrosterone (DHEA)-induced hepatic lesions for the peroxisomal beta-oxidation system enzyme peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (PBE) and its mRNA using SDS-polyacrylamide gel electrophoresis, antibodies and cDNA probe. All 12 neoplastic nodules and nine hepatocellular carcinomas (HCCs) that were analyzed for PBE mRNA by in situ hybridization showed an intense signal comparable to the adjacent non-neoplastic liver. SDS-polyacrylamide gel electrophoresis of postnuclear fractions of six HCC and adjacent liver tissue showed a marked increase in an 80 kDa polypeptide. Immunoblot and Northern blot analysis showed a marked increase in PBE enzyme and PBE mRNA respectively in HCC and adjacent non-neoplastic liver tissue. In control livers (animals not treated with peroxisome proliferators), the levels of PBE enzyme and mRNA were very low or undetectable. The results of this study clearly indicate that peroxisome proliferator (PP)-induced liver lesions express peroxisomal enzymes to the same extent as adjacent liver and that these enzymes are not useful markers for identification of PP-induced lesions.

Levels of dopachrome tautomerase in human melanocytes cultured in vitro.

Several reports have been published about the level of activity and possible functions of dopachrome tautomerase (DCT) in mouse melanoma cells. Data about the levels of this activity in human melanocytes in culture are still scarce, and, as far as we know, a comparison between mouse and human melanocytes, or between normal and malignant melanocytes, has never been published. We have measured the tyrosinase and DCT activities, as well as the melanin content, in mouse Cloudman melanoma cells, two lines of human melanoma, and three lines of normal human melanocytes obtained from fetal skin. Although more cell lines should be tested to draw a general conclusion, our results suggest that normal melanocytes contained much higher tyrosinase activity and melanin content but lower DCT activity than malignant melanocytes. The two lines of human melanoma cells tested had lower levels of DCT activity than Cloudman melanoma cells. Finally, the low level of DCT activity found in normal human melanocytes cultured in vitro cannot be explained by any of the necessary stimulatory factors added to the cell culture media.

CaBP1, a calcium binding protein of the thioredoxin family, is a resident KDEL protein of the ER and not of the intermediate compartment.

A cDNA encoding rat CaBP1 has been isolated and sequenced. The deduced polypeptide chain consists of 440 amino acids including two internal thioredoxin-like domains and a C-terminal KDEL retention/retrieval signal. Regarding the high degree of identity to the hamster protein P5, CaBP1 is considered to be the homologous rat protein. Previous work has suggested that CaBP1 is a resident luminal protein of the intermediate compartment (Schweizer, A., Peter, F., Nguyen Van, P., Söling, H.D. and Hauri, H.P. (1993) Eur. J. Cell Biol. 60, 366-370). Our conclusion that CaBP1 is a resident protein of the endoplasmic reticulum and not of the intermediate compartment is based on three different approaches: subcellular fractionation, indirect immunofluorescence and overexpression of CaBP1. Subcellular fractionation of Vero cells in a velocity controlled step gradient led to copurification of CaBP1-containing vesicles and several marker proteins for the ER including calreticulin and alpha-SSRP. The intermediate compartment, as defined by a monoclonal antibody against the marker protein p53 (ERGIC-53), could be separated from these ER markers. Double immunofluorescence analysed by laser scanning microscopy showed no significant colocalization between CaBP1 and p53, but between CaBP1 and calreticulin. In addition experiments, Vero cells were infected with VSV tsO45. At 15 degrees C the VSV-G protein accumulated in punctuate structures representing the intermediate compartment, while CaBP1 maintained its original reticular localization. Even after high-level overexpression in COS cells, CaBP1 was not detected in the intermediate compartment, but was efficiently retained in the ER as judged by light microscopy.