Clinicopathological features of cytokeratin 5-positive pulmonary adenocarcinoma.

Cytokeratin 5 (CK5) is a marker for pulmonary squamous cell carcinoma; however, CK5 is sometimes present in pulmonary adenocarcinoma (ADC), and there is insufficient information regarding the clinicopathological features of CK5-positive ADC. We aimed to explore the clinicopathological characteristics of CK5-positive ADC using immunohistochemistry. We prepared the following two cohorts: a resected cohort containing 220 resected tumours for primarily studying the detailed morphological characteristics, and a tissue microarray (TMA) cohort containing 337 samples for investigating the associations of CK5 expression with other protein expressions, genetic and prognostic findings. CK5-positive ADC was defined to have ≥ 10% tumour cells and presence of CK5-positive tumour cells in the resected and TMA cohorts, respectively. CK5-positive ADCs were identified in 91 (16.3%) patients in the combined cohort. CK5-positive ADCs had male predominance (P = 0.012), smoking history (P = 0.001), higher stage (P < 0.001), histological high-grade components (P < 0.001), vascular invasion (P < 0.001), mucinous differentiation (P < 0.001), spread through airspaces (P < 0.001), EGFR wild-type (P < 0.001), KRAS mutations (P < 0.001), ALK rearrangement (P < 0.001) and ROS1 rearrangement (P = 0.002). In the resected cohort, more than half the CK5-positive ADCs (19 cases, 65.5%) showed mucinous differentiation; the remaining cases harboured high-grade components. In the TMA cohort, CK5-positive ADCs correlated with TTF-1 negativity (P = 0.002) and MUC5B, MUC5AC and HNF4alpha positivity (P < 0.001, 0.048, < 0.001). Further, CK5-positive ADCs had significantly lower disease-free and overall survival rates than CK5-negative ADCs (P < 0.001 for each). Additionally, multivariate analysis revealed that CK5 expression was an independent poor prognostic factor. CK5-positive ADCs showed aggressive clinical behaviour, with high-grade morphology and mucinous differentiation.

Angiopoietin-like protein 2 promotes chondrogenic differentiation during bone growth as a cartilage matrix factor.

OBJECTIVE: Chondrocyte differentiation is crucial for long bone growth. Many cartilage extracellular matrix (ECM) proteins reportedly contribute to chondrocyte differentiation, indicating that mechanisms underlying chondrocyte differentiation are likely more complex than previously appreciated. Angiopoietin-like protein 2 (ANGPTL2) is a secreted factor normally abundantly produced in mesenchymal lineage cells such as adipocytes and fibroblasts, but its loss contributes to the pathogenesis of lifestyle- or aging-related diseases. However, the function of ANGPTL2 in chondrocytes, which are also differentiated from mesenchymal stem cells, remains unclear. Here, we investigate whether ANGPTL2 is expressed in or functions in chondrocytes. METHODS: First, we evaluated Angptl2 expression during chondrocyte differentiation using chondrogenic ATDC5 cells and wild-type epiphyseal cartilage of newborn mice. We next assessed ANGPTL2 function in chondrogenic differentiation and associated signaling using Angptl2 knockdown ATDC5 cells and Angptl2 knockout mice. RESULTS: ANGPTL2 is expressed in chondrocytes, particularly those located in resting and proliferative zones, and accumulates in ECM surrounding chondrocytes. Interestingly, long bone growth was retarded in Angptl2 knockout mice from neonatal to adult stages via attenuation of chondrocyte differentiation. Both in vivo and in vitro experiments show that changes in ANGPTL2 expression can also alter p38 mitogen-activated protein kinase (MAPK) activity mediated by integrin α5ß1. CONCLUSION: ANGPTL2 contributes to chondrocyte differentiation and subsequent endochondral ossification through α5ß1 integrin and p38 MAPK signaling during bone growth. Our findings provide insight into molecular mechanisms governing communication between chondrocytes and surrounding ECM components in bone growth activities.

Stiripentol for the treatment of super-refractory status epilepticus with cross-sensitivity.

BACKGROUND: Cross-sensitivity of rash has been reported between various antiepileptic drugs (AEDs). However, few studies have determined the frequency and management of cross-sensitivity in patients with super-refractory status epilepticus (SRSE). AIMS OF THE STUDY: To examine the optimal AED for treating SRSE with cross-sensitivity. METHODS: We performed a retrospective review of adult patients with SRSE treated at Nagoya City University Hospital, in which we investigated the frequency of cross-sensitivity among patients with SRSE and their clinical and medical profiles. RESULTS: We identified 10 adult patients with SRSE, 5 of whom had cross-sensitivity. Stiripentol (STP) was administered when previously used AEDs had demonstrated cross-sensitivity and failed to control seizures. After initiation of STP, the dose of general anaesthetics was reduced, and status epilepticus (SE) eventually ceased with co-administered AEDs without additional adverse effects. The mean time to SE cessation after initiation of STP was 30.8 days (range, 18-46 days), mean duration of general anaesthesia was 101.2 days (range, 74-128 days), and mean number of AEDs was 9.0 (range, 6-11). CONCLUSIONS: This study suggests that cross-sensitivity between AEDs is common in adults with SRSE and that STP may be useful for treating SRSE with cross-sensitivity.

Evidence for the role of INSL3 on sperm production in boars by passive immunisation.

Insulin-like factor 3 (INSL3), previously called relaxin-like factor, is essential for foetal testis descent and has been implicated in sperm production in adult males. This study investigated the role of INSL3 in sperm production by examining the effect of neutralising INSL3 by passive immunisation on testicular function and sperm output in boars. Six male Duroc boars were randomly assigned to passive immunisation and control groups (n = 3 each). The immunisation group was intravenously injected with an IgG fraction of anti-INSL3 antibody developed against the B domain of INSL3 at 2-week intervals from 21-40 weeks of age. The control group was treated with normal IgG in the same manner. Antibody administration reduced testis weight and caused a fourfold increase in the frequency of apoptotic germ cells, which was associated with upregulation of the pro-apoptotic caspase 3 and BAX, and downregulation of the anti-apoptotic XIAP and BCL2, and a substantial marked reduction in sperm concentration. Neutralising INSL3 delivered by passive immunisation reduced testis weight and sperm concentration by inducing germ cell apoptosis, suggesting that INSL3 acts as a germ cell survival/anti-apoptotic factor in the maintenance of sperm production.

Formation and evolution of carbonaceous asteroid Ryugu: Direct evidence from returned samples.

Samples of the carbonaceous asteroid Ryugu were brought to Earth by the Hayabusa2 spacecraft. We analyzed 17 Ryugu samples measuring 1 to 8 millimeters. Carbon dioxide-bearing water inclusions are present within a pyrrhotite crystal, indicating that Ryugu's parent asteroid formed in the outer Solar System. The samples contain low abundances of materials that formed at high temperatures, such as chondrules and calcium- and aluminum-rich inclusions. The samples are rich in phyllosilicates and carbonates, which formed through aqueous alteration reactions at low temperature, high pH, and water/rock ratios of <1 (by mass). Less altered fragments contain olivine, pyroxene, amorphous silicates, calcite, and phosphide. Numerical simulations, based on the mineralogical and physical properties of the samples, indicate that Ryugu's parent body formed ~2 million years after the beginning of Solar System formation.

Usefulness of endoscopic retrograde biliary biopsy using large-capacity forceps for extrahepatic biliary strictures: a prospective randomized study.

BACKGROUND AND STUDY AIMS: Endoscopic retrograde biliary biopsy samples are frequently too small and inadequate, which makes histological interpretation difficult. We therefore evaluated the diagnostic usefulness of forceps with a larger-sized cup and compared this with standard forceps for biliary biopsy. PATIENTS AND METHODS: This prospective study included consecutive patients with extrahepatic biliary strictures who underwent retrograde biliary biopsy between March 2005 and March 2006 at the Toho University Ohashi Medical Center. The standard forceps used were 1.8-mm forceps (FB-39Q, Olympus, Tokyo, Japan) and the large-capacity forceps were 2.2-mm forceps (Radial jaw3, Boston Scientific Inc., Natick, Massachusetts, USA). Four randomized biopsy specimens were taken from each patient, two using each type of forceps. RESULTS: A total of 32 patients (30 with malignant biliary strictures and 2 with benign biliary strictures) were enrolled. The median size of the biopsy samples taken using the standard forceps was 0.68 mm (2) and that using the large-capacity forceps was 1.98 mm (2) ( P < 0.0001). Significant differences between the standard forceps and large-capacity forceps were observed in sensitivity (43 % vs. 70 %), adequacy of the specimens, and submucosal tissue sampling rate. CONCLUSIONS: Large-capacity forceps performed better than standard forceps in terms of size, adequacy of the sample, submucosal sampling rate, and detection of neoplasia.

The human DnaJ homologue dj2 facilitates mitochondrial protein import and luciferase refolding.

DnaJ homologues function in cooperation with hsp70 family members in various cellular processes including intracellular protein trafficking and folding. Three human DnaJ homologues present in the cytosol have been identified: dj1 (hsp40/hdj-1), dj2 (HSDJ/hdj-2), and neuronal tissue-specific hsj1. dj1 is thought to be engaged in folding of nascent polypeptides, whereas functions of the other DnaJ homologues remain to be elucidated. To investigate roles of dj2 and dj1, we developed a system of chaperone depletion from and readdition to rabbit reticulocyte lysates. Using this system, we found that heat shock cognate 70 protein (hsc70) and dj2, but not dj1, are involved in mitochondrial import of preornithine transcarbamylase. Bacterial DnaJ could replace mammalian dj2 in mitochondrial protein import. We also tested the effects of these DnaJ homologues on folding of guanidine-denatured firefly luciferase. Unexpectedly, dj2, but not dj1, together with hsc70 refolded the protein efficiently. We propose that dj2 is the functional partner DnaJ homologue of hsc70 in the mammalian cytosol. Bacterial DnaJ protein could replace mammalian dj2 in the refolding of luciferase. Thus, the cytosolic chaperone system for mitochondrial protein import and for protein folding is highly conserved, involving DnaK and DnaJ in bacteria, Ssa1-4p and Ydj1p in yeast, and hsc70 and dj2 in mammals.

Impact of gastro-oesophageal reflux disease symptoms on COPD exacerbation.

BACKGROUND: The association between gastro-oesophageal reflux disease (GORD) and chronic obstructive pulmonary disease (COPD) exacerbation has so far remained unclear. OBJECTIVE: To prospectively establish the clinical significance of GORD symptoms on exacerbation. METHODS: 82 patients with COPD and 40 age matched controls were enrolled in this study. Symptoms were evaluated by a questionnaire using the Frequency Scale for the Symptoms of GORD (FSSG). Patients with COPD were prospectively surveyed for 6 months, and episodes of exacerbation were identified using a diary based on modified Anthonisen's criteria. Exhaled breath condensate (EBC) pH was measured in both groups, and induced sputum was evaluated in patients with COPD. RESULTS: Positive GORD symptoms were reported in 22 (26.8%) patients with COPD and in five (12.5%) controls (p = 0.10). The frequency of exacerbations was significantly associated with the FSSG score (p = 0.03, r = 0.24, 95% CI 0.02 to 0.43). Multiple regression analysis revealed that GORD symptoms were significantly associated with the occurrence of exacerbations (p<0.01; relative risk 6.55, 95% CI 1.86 to 23.11). EBC pH was inversely correlated with FSSG score in both groups (p = 0.01, r = -0.37, 95% CI -0.55 to -0.14 in patients with COPD, and p<0.01, r = -0.45, 95% CI -0.67 to -0.16 in control subjects). CONCLUSIONS: GORD symptoms were identified as an important factor associated with COPD exacerbation.

Applicability of DPI formulations for novel neurokinin receptor antagonist.

A novel triple neurokinin receptor antagonist (TNRA) could have pharmaceutical efficacy for asthma and/or chronic obstructive pulmonary disease. TNRA is potentially developed as inhalation medicine. The aim of this investigation was to evaluate the applicability of dry powder inhaler (DPI) formulation for TNRA. DPI formulation containing lactose was used for this feasibility study. Mechanofusion process for surface modification was applied on lactose particles to prepare four different DPI formulations. The mixture of TNRA and lactose was administered to rats intratracheally using an insufflator. The deposition pattern and blood concentration profile of TNRA were evaluated. Although there was no significant difference in deposition on deep lungs between the four formulations, DPI formulations containing mechanofusion-processed lactose showed longer T(max) and t(1/2) and higher AUC(0-infinity) and MRT compared to that containing intact lactose. On the other hand, the contact angle measurement showed that the mechanofusion process decreased the polar part of the surface energy of the lactose. Therefore, the prolongation of the wetting of the formulated powder mixture seemed to delay the dissolution of TNRA deposited in respiratory tract. It was concluded that DPI formulation containing mechanofusion-processed lactose could be suitable for inhalation of TNRA.

Thermal and impact histories of 25143 Itokawa recorded in Hayabusa particles.

Understanding the origin and evolution of near-Earth asteroids (NEAs) is an issue of scientific interest and practical importance because NEAs are potentially hazardous to the Earth. However, when and how NEAs formed and their evolutionary history remain enigmas. Here, we report the U-Pb systematics of Itokawa particles for the first time. Ion microprobe analyses of seven phosphate grains from a single particle provide an isochron age of 4.64 ± 0.18 billion years (1σ). This ancient phosphate age is thought to represent the thermal metamorphism of Itokawa's parent body, which is identical to that of typical LL chondrites. In addition, the incorporation of other particles suggests that a significant shock event might have occurred 1.51 ± 0.85 billion years ago (1σ), which is significantly different from the shock ages of 4.2 billion years of the majority of shocked LL chondrites and similar to that of the Chelyabinsk meteorite. Combining these data with recent Ar-Ar studies on particles from a different landing site, we conclude that a globally intense impact, possibly a catastrophic event, occurred ca. 1.4 Ga ago. This conclusion enables us to establish constraints on the timescale of asteroid disruption frequency, the validity of the crater chronology and the mean lifetime of small NEAs.

Infection of human synovial cells by human T cell lymphotropic virus type I. Proliferation and granulocyte/macrophage colony-stimulating factor production by synovial cells.

The present study was performed to clarify the relationship between human T cell lymphotropic virus type I (HTLV-I) infection and chronic inflammatory arthropathy. To determine the ability of HTLV-I to infect synovial cells and the effect on synovial cell proliferation, synovial cells were cocultured with the HTLV-I-producing T cell lines (MT-2 or HCT-1). After coculture with HTLV-I-infected T cells, the synovial cells expressed HTLV-I-specific core antigens, and HTLV-I proviral DNA was detected from the synovial cells by polymerase chain reaction. These cocultured synovial cells with HTLV-I-infected T cells proliferated more actively than the synovial cells cocultured with uninfected T cells. This stimulatory effect of HTLV-I-infected T cells on synovial cell proliferation seems necessary to contact each other. After being cocultured with MT-2 cells, synovial cells proliferated more actively than control cells even after several passages. Furthermore, HTLV-I-infected synovial cells produced significant amounts of granulocyte/macrophage colony-stimulating factor. These results suggest that HTLV-I can infect synovial cells, resulting their active proliferation and may be involved in the pathogenesis of proliferative synovitis similar to that found in rheumatoid arthritis.

Absence of IgD-CD27(+) memory B cell population in X-linked hyper-IgM syndrome.

The present study analyzed peripheral blood B cell populations separated by IgD and CD27 expression in six males with X-linked hyper-IgM syndrome (XHIM). Costimulation of mononuclear cells from most of the patients induced no to low levels of class switching from IgM to IgG and IgA with Staphylococcus aureus Cowan strain (SAC) plus IL-2 or anti-CD40 mAb (anti-CD40) plus IL-10. Measurable levels of IgE were secreted in some of the patients after stimulation with anti-CD40 plus IL-4. Costimulation with SAC plus IL-2 plus anti-CD40 plus IL-10 yielded secretion of significant levels of IgG in addition to IgM, but not IgA. The most striking finding was that peripheral blood B cells from all of the six patients were composed of only IgD+ CD27(-) and IgD+ CD27(+) B cells; IgD- CD27(+) memory B cells were greatly decreased. IgD+ CD27(+) B cells from an XHIM patient produced IgM predominantly. Our data indicate that the low response of IgG production in XHIM patients is due to reduced numbers of IgD- CD27(+) memory B cells. However, the IgG production can be induced by stimulation of immunoglobulin receptors and CD40 in cooperation with such cytokines as IL-2 and IL-10 in vitro.

Identification and functional analysis of human Tom22 for protein import into mitochondria.

Mitochondria have a receptor complex in the outer membrane which recognizes and translocates mitochondrial proteins synthesized in the cytosol. We report here the identification and functional analysis of human Tom22 (hTom22). hTom22 has an N-terminal negatively charged region exposed to the cytosol, a putative transmembrane region, and a C-terminal intermembrane space region with little negative charge. Tom22 forms a complex with Tom20, and its cytosolic domain functions as an import receptor as in fungi. An import inhibition assay, using pre-ornithine transcarbamylase (pOTC) derivatives and a series of hTom22 deletion mutants, showed that the C-terminal segment of the cytosolic domain is important for presequence binding, whereas the N-terminal domain is important for binding to the mature portion of pOTC. No evidence for pOTC interaction with the Tom22 intermembrane space domain was obtained. Binding studies revealed that the presequence is critical for pOTC binding to Tom20, whereas both the presequence and mature portion are important for binding to Tom22. A cell-free immunoprecipitation assay indicated that an internal segment of the Tom22 cytosolic domain is important for interaction with Tom20.

Role of heat shock cognate 70 protein in import of ornithine transcarbamylase precursor into mammalian mitochondria.

The roles of the 70-kDa cytosolic heat shock protein (hsp70) in import of precursor proteins into the mitochondria were postulated to be related to (i) unfolding of precursor proteins in the cytosol, (ii) maintenance of the import-competent state, and (iii) unfolding and transport of precursor proteins through contact sites, in cooperation with matrix hsp70. We examined roles of cytosolic hsp70 family members in import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria, using an in vitro import system and antibodies against hsp70. Immunoblot analysis using an hsc70 (70-kDa heat shock cognate protein)-specific monoclonal antibody and a polyclonal antibody that reacts with both hsc70 and hsp70 showed that hsc70 is the only or major form of hsp70 family members in the rabbit reticulocyte lysate. The hsc70 antibody did not inhibit pOTC import when added prior to import assay. However, when pOTC was synthesized in the presence of the antibody and then subjected to import assay, pOTC import was markedly decreased. pOTC import was also decreased when the precursor was synthesized in the lysate depleted for hsc70 by treatment with hsc70 antibody-conjugated Sepharose. This reduction was almost completely restored by readdition of purified mouse hsc70 during pOTC synthesis. The readdition of hsc70 after pOTC synthesis and only during the import assay was not effective. Thus, once import competence of pOTC was lost, hsc70 was ineffective for restoration. Newly synthesized pOTC lost import competence in the absence of hsc70 somewhat more rapidly than in its presence. These results indicate that hsc70 is required during pOTC synthesis and not during import into the mitochondria. hsc70 presumably binds to pOTC polypeptide and maintains it in an import-competent form.

The requirement of heat shock cognate 70 protein for mitochondrial import varies among precursor proteins and depends on precursor length.

The cytosolic heat shock cognate 70-kDa protein (hsc70) is required for efficient import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria (K. Terada, K. Ohtsuka, N. Imamoto, Y. Yoneda, and M. Mori, Mol. Cell. Biol. 15:3708-3713, 1995). The requirement of hsc70 for mitochondrial import of various precursor proteins and truncated pOTCs was studied by using an in vitro translation import system in which hsc70 was completely depleted. hsc70-dependent import of pOTC was about 60% of the total import, while import of the aspartate aminotransferase precursor, the serine:pyruvate aminotransferase precursor, and 3-oxoacyl coenzyme A thiolase was about 50, 30, and 0%, respectively. The subunit sizes of these four precursor proteins were 40 to 47 kDa. When pOTC was serially truncated from the COOH terminal, the hsc70 requirement decreased gradually and was not evident for the shortest truncated pOTCs of 90 and 72 residues. These truncated pOTCs were imported and proteolytically processed rapidly in 0.5 to 2 min at 25 degrees C, and the processed mature portions and the presequence portion were rapidly degraded. Sucrose gradient centrifugation analysis followed by import assay showed that pOTC synthesized in rabbit reticulocyte lysate forms an import-competent complex of about 11S in an hsc70-dependent manner. S values of import-competent forms of aspartate aminotransferase precursor, serine:pyruvate aminotransferase precursor, and 3-oxoacyl coenzyme A thiolase were 9S, 9S, and 4S, respectively. Thus, the S value decreased as the hsc70 dependency decreased. Precursor proteins were coimmunoprecipitated from the reticulocyte lysate containing the newly synthesized precursor proteins with an hsc70 antibody. The amount of coimmunoprecipitated proteins was much larger in the absence of ATP than in its presence. Among the four precursor proteins, the amount of coimmunoprecipitated protein decreased as the hsc70 dependency decreased.

Non-destructive elemental analysis of a carbonaceous chondrite with direct current Muon beam at MuSIC.

Electron- or X-ray-induced characteristic X-ray analysis has been widely used to determine chemical compositions of materials in vast research fields. In recent years, analysis of characteristic X-rays from muonic atoms, in which a muon is captured, has attracted attention because both a muon beam and a muon-induced characteristic X-ray have high transmission abilities. Here we report the first non-destructive elemental analysis of a carbonaceous chondrite using one of the world-leading intense direct current muon beam source (MuSIC; MUon Science Innovative Channel). We successfully detected characteristic muonic X-rays of Mg, Si, Fe, O, S and C from Jbilet Winselwan CM chondrite, of which carbon content is about 2 wt%, and the obtained elemental abundance pattern was consistent with that of CM chondrites. Because of its high sensitivity to carbon, non-destructive elemental analysis with a muon beam can be a novel powerful tool to characterize future retuned samples from carbonaceous asteroids.

Copper-transporting P-type adenosine triphosphatase (ATP7B) is associated with cisplatin resistance.

The accumulation of cisplatin is decreased in many cisplatin-resistant cell lines, and an active efflux pump for cisplatin exists in some of them, but it has not yet been identified. In this study, we transfected the copper-transporting P-type ATPase cDNA (ATP7B) into human epidermoid carcinoma KB-3-1 cells. The transfectant, KB/WD cell line, which overexpressed the P-type ATPase, ATP7B, was resistant to both cisplatin (8.9-fold) and copper (2.0-fold). The accumulation of cisplatin in KB/WD cells was lower than in mock-transfected KB/CV cells, and the efflux of cisplatin from KB/WD cells was enhanced compared with KB/CV cells. KB/WD cells were sensitive to other heavy metals, such as antimony, arsenate, arsenite, cadmium, and cobalt. ATP7B was overexpressed in cisplatin-resistant prostate carcinoma PC-5 cells but not in the parental PC-3 cells and the revertant PC-5R cells. ATP7B may be involved in cisplatin resistance in some tumors.

Magnetic separation of general solid particles realised by a permanent magnet.

Most existing solids are categorised as diamagnetic or weak paramagnetic materials. The possibility of magnetic motion has not been intensively considered for these materials. Here, we demonstrate for the first time that ensembles of heterogeneous particles (diamagnetic bismuth, diamond and graphite particles, as well as two paramagnetic olivines) can be dynamically separated into five fractions by the low field produced by neodymium (NdFeB) magnets during short-duration microgravity (µg). This result is in contrast to the generally accepted notion that ordinary solid materials are magnetically inert. The materials of the separated particles are identified by their magnetic susceptibility (χ), which is determined from the translating velocity. The potential of this approach as an analytical technique is comparable to that of chromatography separation because the extraction of new solid phases from a heterogeneous grain ensemble will lead to important discoveries about inorganic materials. The method is applicable for the separation of the precious samples such as lunar soils and/or the Hayabusa particles recovered from the asteroids, because even micron-order grains can be thoroughly separated without sample-loss.

Copper incorporation into ceruloplasmin in rat livers.

Ceruloplasmin, a blue copper oxidase found in plasma, is synthesized in hepatocytes as a single polypeptide chain consisting of a 19 amino acid leader peptide plus 1046 amino acids of mature protein (132 kDa). Holoceruloplasmin is secreted into the plasma with 6-7 atoms of copper bound per molecule. In this study we identified apo- and holoceruloplasmin and examined the mechanism of copper incorporation during ceruloplasmin biosynthesis using the Long-Evans Cinnamon (LEC) rat which does not incorporate copper into newly synthesized ceruloplasmin. We followed the conversion from ceruloplasmin precursor (with little or no carbohydrate) to the larger product (after carbohydrate addition), which occurred in the secretory compartments of hepatocytes, by native gel electrophoresis. We found that copper accumulates in the hepatocellular Golgi apparatus of LEC rats due to a disorder in the process of copper incorporation. The data indicate that copper is incorporated into ceruloplasmin late in the course of its transport through the secretory compartments.