The KANNO blood group system.

The KANNO blood group system (International Society of Blood Transfusion [ISBT] 037) includes one high-prevalence antigen, KANNO1, across ethnic groups. Sporadic KANNO1- cases among East and South Asians are theoretically estimated by the DNA database library. Anti-KANNO1 has been found most often among Japanese women with current or prior pregnancy. Thus far, there are no reported cases of hemolytic transfusion reaction or hemolytic disease of the fetus and newborn due to anti-KANNO1.

Bioaugmentation as a green technology for hydrocarbon pollution remediation. Problems and prospects.

Environmental pollution mitigation measure involving bioremediation technology is a sustainable intervention for a greener ecosystem biorecovery, especially the obnoxious hydrocarbons, xenobiotics, and other environmental pollutants induced by anthropogenic stressors. Several successful case studies have provided evidence to this paradigm including the putative adoption that the technology is eco-friendly, cost-effective, and shows a high tendency for total contaminants mineralization into innocuous bye-products. The present review reports advances in bioremediation, types, and strategies conventionally adopted in contaminant clean-up. It identified that natural attenuation and biostimulation are faced with notable limitations including the poor remedial outcome under the natural attenuation system and the residual contamination occasion following a biostimulation operation. It remarks that the use of genetically engineered microorganisms shows a potentially promising insight as a prudent remedial approach but is currently challenged by few ethical restrictions and the rural unavailability of the technology. It underscores that bioaugmentation, particularly the use of high cell density assemblages referred to as microbial consortia possess promising remedial prospects thus offers a more sustainable environmental security. The authors, therefore, recommend bioaugmentation for large scale contaminated sites in regions where environmental degradation is commonplace.

An anatomic study of the accessory anterolateral talar facet.

BACKGROUND: A small accessory facet with articular surface morphology is occasionally seen on the talus, bordering on the lateral end of the sinus tarsi. This facet has been named the accessory anterolateral talar facet. However, few anatomical studies have addressed this facet. Here we present the precise morphology of accessory anterolateral talar facet with emphasis on anatomical correlation between the presence of this facet and the angle of the infero-lateral surface of the talus (talar infero-lateral surface - TILS angle). MATERIALS AND METHODS: A total of 22 (11 male, 11 female) adult cadavers with no known pathological conditions in the talocalcaneal joints were examined during educational dissection at Nagoya City University Medical School in 2013. After exclusion of 1 joint due to the poor condition of the talus, 43 talus (22 right, 21 left) were analysed. We judged the presence of the accessory anterolateral talar facet and measured TILS angle. We performed statistical analysis on the point of laterality, gender difference and the difference in the TILS angles in tali with or without the accessory anterolateral talar facets. RESULTS: An accessory anterolateral talar facet was identified in 11 (26%) of the 43 specimens. Of the 21 cadavers with paired talar specimens, 5 displayed the facet bilaterally. CONCLUSIONS: There was no sex difference and no significant laterality, however we found that TILS angle was significantly larger in accessory anterolateral talar facet positive samples than in negative ones.

Morphological characteristics of the deep layer of articularis genus muscle.

BACKGROUND: The articularis genus muscle pulls the suprapatellar pouch upwards when the knee joint is extended, preventing mechanical impingement of the joint capsule which theoretically could cause anterior knee pain. However, few anatomical studies have addressed this muscle. Here we present the precise morphology of articularis genus. MATERIALS AND METHODS: A total of 22 (13 male and 9 female) adult cadavers with no pathological conditions in the knee joints were examined during educational dissection at Nagoya City University Medical School in 2012. After exclusion of 4 joints due to their flexion contracture, 40 knee joints (18 right and 22 left) were analysed. We performed statistical analysis on anatomical laterality and the difference of sizes among lateral, medial and central branches and studied the correlation of the length and area of the articularis genus muscle to the lengthand cross-section area of the femur. RESULTS AND CONCLUSIONS: The average number of branches of the deep layer of the articularis genus muscle was 2.7 ± 0.5, the mean length of all brancheswas 5.4 ± 1.3 cm and the mean area of all branches was 5.5 ± 2.6 cm². There was no significant correlation between the length and area of the articularis genus muscle to the length and cross-section area of the femur. There was no significant laterality in central, medial and lateral branches; however we found that the medial branch was statistically longer and larger than the lateral branchon either knee. This could be contributing to prevention of lateral dislocation of the patella.

Patients' preference for number of embryos transferred during IVF/ICSI: a Nigerian experience.

BACKGROUND: The Human Fertilization and Embryology Authority is considering limiting the number of embryos that can be transferred to single embryo per cycle as has been done in several European countries, with the aim of reducing the rate of multiple pregnancies and its attendant complications following in vitro fertilization (IVF) / Intracytoplasmic sperm injection (ICSI). OBJECTIVE: To determine the number of embryos patients' attending a fertility clinic in Nigeria, would prefer transferred during IVF/ICSI. MATERIALS AND METHODS: Fifty four consecutive female patients who underwent IVF/ICSI procedures between May 2006 and April 2007 at the Port Harcourt Fertility Centre, Rivers State were interviewed using structured questionnaires. They were informed of all the obstetric and perinatal complications of multiple pregnancies and the advantages and trend towards single embryo transfer and then asked to choose the number of embryos (one, two or three) they would prefer transferred assuming similar implantation rates. Each respondent was allowed to give reason(s) for their choice. DESIGN: Prospective, descriptive study. RESULTS: Fifty one (94.4%) of the respondents preferred the transfer of multiple (2 or 3) embryos. Only three (5.6%) patients opted for single embryo transfer. Majority of the patients (31 or 60.8%) preferred multiple embryo transfer because of their desire for twins while twenty (39.2%) cited cost of IVF as their reason. Fifteen (29.4%) patients saw multiple pregnancies as a compensation for their long periods of infertility. CONCLUSION: With the desire for twins and high poverty level in Nigeria, a policy of single embryo transfer might be difficult to implement. Health economic studies would be required to determine if the accumulative cost of taking care of twins/triplets is less, equal or outweighs the cost of several single embryo transfers.

Association of the tyrosine phosphorylated epidermal growth factor receptor with a 55-kD tyrosine phosphorylated protein at the cell surface and in endosomes.

After the intraportal injection of EGF, the EGF receptor (EGFR) is rapidly internalized into hepatic endosomes where it remains largely receptor bound (Lai et al., 1989. J. Cell Biol. 109:2751-2760). In the present study, we evaluated the phosphotyrosine content of EGFRs at the cell surface and in endosomes in order to assess the consequences of internalization. Quantitative estimates of specific radioactivity of the EGFR in these two compartments revealed that tyrosine phosphorylation of the EGFR was observed at the cell surface within 30 s of ligand administration. However, the EGFR was also highly phosphorylated in endosomes reaching levels of tyrosine phosphorylation significantly higher than those of the cell surface receptor at 5 and 15 min after EGF injection. A 55-kD tyrosine phosphorylated polypeptide (pyp55) was observed in association with the EGFR at the cell surface within 30 s of EGF injection. The protein was also found in association with the EGFR in endosomes as evidenced by coprecipitation studies using a mAb to the EGFR as well as by coelution with the EGR in gel permeation chromatography. Limited proteolysis of isolated endosomes indicated that the tyrosine phosphorylated domains of the EGFR and associated pyp55 were cytosolically oriented while internalized EGF was intraluminal. The identification of pyp55 in association with EGFR in both hepatic plasma membranes and endosomes may be relevant to EGFR function and/or trafficking of the EGFR.

Ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo.

EGF receptor internalization, recycling,a nd downregulation were evaluated in liver parenchyma as a function of increasing doses of injected EGF. The effect of ligand occupancy in vivo on the kinetics and extent of internalization was studied with changes in the receptor content of isolated plasmalemma and endosome fractions evaluated by direct binding, Scatchard analysis, and Western blotting. For all doses of injected EGF, receptor was lost from the plasmalemma and accumulated in endosomes in a time- and dose-dependent fashion. However, at doses of injected EGF equivalent to less than or equal to 50% surface receptor occupancy (i.e., less than or equal to 1 microgram/100 g body weight), receptor levels returned by 120 min to initial values. This return was resistant to cycloheximide and therefore did not represent newly synthesized receptor. Neither was the return due to replenishment by an intracellular pool of low-affinity receptors as such a pool could not be detected by Scatchard analysis or Western blotting. Therefore, receptor return was due to the recycling of previously internalized receptor. At doses of injected EGF greater than 50% receptor occupancy, net receptor loss-i.e., downregulation-was observed by evaluating the receptor content of total particulate fractions of liver homogenates. At the higher saturating doses of injected EGF (5 and 10 micrograms/100 g body weight), the majority of surface receptor content was lost by 15 min and remained low for at least an additional 105 min. As the kinetics of ligand clearance from the circulation and liver parenchyma were similar for all doses of EGF injected, then the ligand-mediated regulation of surface receptor content and downregulation were not a result of a prolonged temporal interaction of ligand with receptor. Rather, the phenomena must be a consequence of the absolute concentrations of EGF interacting with receptor at the cell surface and/or in endosomes.

Assessment of haemophilia treatment practice pattern in Japan.

The current status of replacement therapy for haemophilia and the role played by nurses in Japan were investigated at 16 facilities (17 specialties) each providing care to 20 or more haemophilia A or B patients without inhibitor. The questionnaire was mailed to the nurse or physician in charge of haemophilia at each facility in August 2006, asking the nurse to fill in the questionnaire. Responses were collected on 1318 patients (haemophilia A: 1078 patients; haemophilia B: 240 patients). About 70% of these patients were reported to be severe haemophilia A or B. Overall, 26% were receiving regular prophylaxis while 74% received on-demand therapy with or without temporary prophylaxis before special events. The percentage of patients receiving primary prophylaxis was only 2%. The percentage of adherence to prophylaxis decreased with age (lowest at age 19-29) but this percentage for each age group in Japan was higher than that in the western countries. Of the nurses working at the facilities surveyed, 88% considered prophylaxis as an optimal therapy for severe haemophilia patients, although the percentage of patients receiving prophylaxis for the entire population surveyed was lower than that in the western countries. The main factor precluding introduction of prophylaxis was 'difficulty in venous access' for infants and small children. On the other hand, 'unwillingness of family members' and 'poor adherence' were the main factors precluding introduction of this therapy for those aged over 6 years.

The effect of endometrial thickness on in vitro fertilization (IVF)-embryo transfer/intracytoplasmic sperm injection (ICSI) outcome.

The value of measuring the endometrial thickness and studying the endometrial receptivity in the context of assisted conception remains a contentious issue. A prospective analysis was carried out to determine the effect of endometrial thickness on IVF - embryo transfer/ICSI outcome in dedicated Assisted Reproductive Technology (ART) units in Abuja and Rivers State, Nigeria. Two hundred and fifty one patients who met the inclusion criteria were analysed. They were grouped on the basis of endometrial thickness into 3 groups; <7 mm, 7 - 14 mm and >14 mm. The main outcome measure was clinical pregnancy. There were significantly more pregnancies in the 7 - 14 mm endometrial thickness group compared to the <7 mm and >14 mm groups, p=0.004 and p<0.0001 respectively. The findings suggest that following IVF/ICSI, significantly more pregnancies occurred when the endometrial thickness was between 7 and 14 mm.

Aberrant expression of photoreceptor-specific calcium-binding protein (recoverin) in cancer cell lines.

Cancer-associated retinopathy (CAR) is an ocular manifestation of a paraneoplastic syndrome whereby immunological reactions to retinal antigens aberrantly expressed in tumor cells lead to the degeneration of retinal photoreceptor cells. In our previous study (H. Ohguro et al., Invest. Ophthalmol. Vis. Sci., 40: 82-89, 1999), recoverin, a retina-specific calcium-binding protein, and heat shock cognate protein 70 (hsc 70) were identified as autoantigens recognized by sera from patients with CAR. Therefore, we suggested that autoimmune reactions against both recoverin and hsc 70 might be involved in the pathogenesis of CAR. To elucidate the initial step of the molecular pathology of CAR, we examined the expression of recoverin and hsc 70 by reverse transcription-PCR and Western blot using cell lines of several kinds of cancers, including lung small cell carcinoma, lung adenocarcinoma, gastric cancer, pancreatic cancer, breast cancer, uterine cervical cancer, endometrial cancer, and leukemia. Recoverin was expressed in 21 of the 31 cancer cell lines. The expression levels of hsc 70 were significantly higher in cancer cell lines than in noncancerous cell lines. However, no difference in the expression levels of hsc 70 was observed between recoverin-positive and -negative cell lines. Immunofluorescence labeling by the affinity-purified recoverin antibody revealed the immunoreactivity to recoverin as a granular pattern within the cancer cells. Lung adenocarcinoma A549 cells, which did not express recoverin, exhibited a significant reduction in cell proliferation upon transfection with human recoverin cDNA. Taken together, our present data suggest that the retina-specific calcium-binding protein recoverin is expressed in more than 50% of a variety of cancer cells and may play a significant role in the cell proliferation of these tumor cells.

Phosphatidic acid phosphatase from mammalian tissues: discovery of channel-like proteins with unexpected functions.

Phosphatidic acid phosphatase (PAP) has long been known as a key enzyme involved in both glycerolipid biosynthesis and cellular signal transduction. The cDNA cloning of a plasma membrane-bound type 2 PAP has revealed the existence of a novel glycoprotein with six transmembrane domains. The type 2 PAP now represents an enzyme family consisting of Drosophila Wunen and rat Dri 42, which participate in germ cell migration and epithelial differentiation, respectively. Such novel functions of the type 2 PAP suggest the unexpected importance of lipids and/or their metabolic enzymes.

New strategy of therapy for functional dyspepsia using famotidine, mosapride and amitriptyline.

BACKGROUND: In functional gastrointestinal (GI) disorders including functional dyspepsia (FD) and irritable bowel syndrome (IBS), there might be no small extent of contributions of psychosomatic factors. As a therapy for IBS patients, the effectiveness of antidepressants has been reported. AIM: In this study, we evaluated the efficacy of H2-receptor antagonist (famotidine) and 5-HT4 receptor agonist (mosapride citrate). In addition, the effect of antidepressants was assessed as the second-step therapy. METHODS: Patients complaining upper GI symptoms were diagnosed as FD excluding organic diseases. Randomized patients received 20 mg/day of famotidine or 15 mg/day of mosapride citrate for 4 weeks and the efficacy was compared between the two groups based on a 10-point visual analogue scale. When symptoms were not relieved (score improvement 0-2 points), patients received amitriptyline (30 mg/day) or no medication for 4 weeks randomly. Patients who had depression in psychological test (SDS) were omitted. RESULTS: As the first-step therapy, both famotidine and mosapride showed beneficial effects regardless of FD subtypes, age and gender. The efficacy of these two drugs in relieving FD symptoms was not significantly different. In patients who failed in the first-step therapy, amitriptyline showed beneficial effects. CONCLUSIONS: These findings might be clinically important in view of the efficient relief of symptoms in FD patients.

Oculocutaneous albinism types 1 and 3 are ER retention diseases: mutation of tyrosinase or Tyrp1 can affect the processing of both mutant and wild-type proteins.

Various types of oculocutaneous albinism (OCA) are associated with reduced pigmentation in the skin, hair, and eyes that results from mutations in genes involved in melanin synthesis. Immortal mouse melanocyte lines (melan-a, melan-b, and melan-c) provide opportune models with which to investigate the etiology of two different types of OCA (types I and III), which arise from mutations in Tyr and Tyrp1, respectively. We compared intracellular processing, sorting, and degradation of tyrosinase and Tyrp1, and the effects on their catalytic function and melanin synthesis, in these wild-type and mutant melanocytes. A mutation in either Tyr or Tyrp1 increased the time of association of tyrosinase and Tyrp1 with calnexin and Bip, which in turn resulted in the retention of these mutant products in the ER. A mutation in either gene selectively enhanced the duration and efficiency of chaperone interactions (even with the wild-type protein in the mutant melanocytes) and markedly slowed their transport to melanosomes. These results show that OCA1 and OCA3 are (in some cases, at least) ER retention diseases wherein a mutation in one melanogenic protein affects the maturation and stability of the other in the melanogenic pathway.

Translocation of diacylglycerol kinase alpha to the nuclear matrix of rat thymocytes and peripheral T-lymphocytes.

The cytosolic alpha-diacylglycerol kinase (DGK) was translocated to and tightly associated with the nuclear matrix when rat thymocytes and peripheral T-lymphocytes were stimulated with concanavalin A or anti-T-cell receptor antibody. This translocation occurred rather slowly and was completed in 3-4 h after cell stimulation. We also detected significant accumulation of nuclear phosphatidic acid interpreted as being formed by the translocated enzyme. The enzyme translocation is not directly linked to phosphoinositide turnover and protein phosphorylation, since phorbol myristate acetate and calcium ionophore did not affect the cellular DGK alpha and since we detected no covalent modification of the enzyme molecule. Although the mechanisms underlying the enzyme translocation remain unknown, our results indicate that DGK alpha participates in nuclear phospholipid metabolism occurring at the intermediate stage of lymphocyte activation.

Features of replicative senescence induced by direct addition of antennapedia-p16INK4A fusion protein to human diploid fibroblasts.

The p16INK4A cyclin-dependent kinase (Cdk) inhibitor is now recognized as a major tumor suppressor that is inactivated by a variety of mechanisms in a wide range of human cancers. It is also implicated in the mechanisms underlying replicative senescence since p16INK4A RNA and protein accumulate as cells approach their proscribed limit of population doublings in tissue culture. To obtain further evidence of its role in senescence, we have sought ways of overexpressing p16INK4A in primary human diploid fibroblasts (HDF). To circumvent the low transfection efficiency of primary cells we have exploited a recombinant form of the full-length p16INK4A protein fused to a 16 amino acid peptide from the Drosophila antennapedia protein. This peptide has the capacity to cross both cytoplasmic and nuclear membranes allowing the direct introduction of the active protein to primary cells. Here, we show that antennapedia-tagged wild-type p16INK4A protein, but not a functionally compromised tumor-specific variant, causes G1 arrest in early passage HDFs by inhibiting the phosphorylation of the retinoblastoma protein. Significantly, the arrested cells display several phenotypic features that are considered characteristic of senescent cells. These data support a role for p16INK4A in replicative senescence and raise the possibility of using the antennapedia-tagged protein therapeutically.

Inductive effect on hepatic enzymes and toxicity of congeners of PCBs and PCDFs.

The present paper describes a marked induction of liver microsomal cytochrome P-450 and cytosolic DT-diaphorase to cause possible disorder of steroid homeostasis and promotion of carcinogenicity of 4-nitroquinoline N-oxide (4-NQO) in rats by pretreatment with 3,4,5,3',4'-pentachlorobiphenyl (PenCB) or 2,3,4,7,8-pentachlorodibenzofuran (PenCDF). The animals were sacrificed 5 days after the pretreatment. These induction experiments showed that 7 alpha-hydroxylation of both progesterone and testosterone in liver microsomes was selectively increased to a great extent, but hydroxylations at the 2 alpha-, 6 beta- and 16 alpha-positions were depressed, together with 5 alpha-reduction. From the same microsomes, three of the strongly induced P-450 isozymes, i.e., high- and low-spin P-448s and P-452, were purified. The last isozyme was most responsible for 7 alpha-hydroxylation of testosterone. The pretreatment, also increased activity of DT-diaphorase and reduction of 4-NQO about 10-fold in liver 9000g supernatants. This reduction of 4-NQO was solely catalyzed by DT-diaphorase and the only product was 4-hydroxylaminoquinoline N-oxide, a proximate carcinogen, indicating that the pretreatment strongly increased production of a proximate carcinogen from 4-NQO. Such an enhancement of the metabolic activation of 4-NQO by the pretreatment was also observed to some extent in the lung and the skin. Persistency of PenCB and PenCDF in the liver of rats was also discussed.

5'-Nucleotidase in rat liver lysosomes.

5'-Nucleotidase was found in purified rat liver tritosomes. When tritosomes were subfractionated into the membrane and soluble contents fractions, 73% of the total 5'-nucleotidase activity was found in the membrane fraction and 24% in the soluble contents fraction. Immunoblotting using specific polyclonal antibodies against the rat liver plasma membrane 5'-nucleotidase showed that the mobilities on SDS-polyacrylamide gel electrophoresis of both 5'-nucleotidases in the membrane and contents fractions were identical to that of the enzyme in the plasma membranes (Mr = 72,000). 5'-Nucleotidases in the membrane and contents fractions were sensitive to neuraminidase and converted into a form that was 4 kDa smaller after digestion, as observed in the case of plasma membrane enzyme. 5'-Nucleotidases, both from the membrane and contents fractions, were purified using immunoaffinity chromatography, and the isoelectric points, heat stability, and oligomeric structure of the purified enzymes were compared. Isoelectric focusing and the heat stability test indicated the resemblance of the soluble enzyme to the membrane-bound enzyme. However, the membrane-bound enzyme aggregated in the absence of Triton X-100, whereas the soluble enzyme behaved as a dimer. The topography of 5'-nucleotidase in the tritosomal membranes was studied using antibodies against 5'-nucleotidase and neuraminidase treatment. The inhibition of 5'-nucleotidase were not observed in the intact tritosomal fraction until the tritosomes had been disrupted by osmotic shock. These results show that the active sites and the oligosaccharide chains of 5'-nucleotidase are located on the inside surface of the tritosomal membranes.

Cell surface activities of the human type 2b phosphatidic acid phosphatase.

Several isozymes of mammalian type 2, Mg(2+)-independent phosphatidic acid phosphatase (PAP-2) have recently been cloned, and they are predicted to have their catalytic sites exposed at the cell surface membranes. We investigated the mode of utilization of extracellular lipid substrates by the human PAP-2b expressed in HEK293 cells as a green fluorescent fusion protein. We first confirmed the plasma membrane localization of the expressed PAP-2b. PAP-2b actively hydrolyzed exogenously added lysophosphatidic acid and short-chain phosphatidic acid. In the case of dephosphorylation of lysophosphatidic acid, the reaction products, including inorganic phosphate and monoacylglycerol, were recovered exclusively in the extracellular medium. Interestingly, PAP-2b exhibited negligible activities toward long-chain phosphatidic acid either exogenously when added or generated within the membranes by treating the cells with bacterial phospholipase D. These findings indicate that PAP-2b acts at the outer leaflet of cell surface bilayers and can account for the ecto-PAP activities previously described for various types of cells.

Comparison of DT-diaphorases purified from the liver cytosol of untreated, and 3,4,5,3',4'-pentachlorobiphenyl- and 3-methylcholanthrene-treated rats.

DT-Diaphorase purified from the liver cytosol of rats treated with a highly toxic PCB congener, 3,4,5,3',4'-pentachlorobiphenyl (PenCB), was compared to those from 3-methylcholanthrene (MC)-treated and untreated rats. Treatments with PenCB and MC resulted in about 8- and 7-fold increases of cytosolic DT-diaphorase activity, respectively. Purification of the enzyme preparations from untreated, and PenCB- and MC-treated rats were conducted by using DE-52, DEAE-Sephadex A-50, hydroxylapatite, and Bio-Gel P-150 column chromatographies. Both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that all of the final preparations from the three origins were homogeneous and had the same molecular weight of 59,000, consisting of two subunits with molecular weights of 30,000. Further studies on amino acid composition, Km value, optimum pH, and catalytic activities for various substrates also indicated that both PenCB- and MC-inducible DT-diaphorases were identical with that from the untreated rats. All three DT-diaphorases contained about 2 mol of FAD per mol of enzyme. Partial digestion of the enzymes by trypsin and subsequent analysis by HPLC revealed that the three preparations were indistinguishable. The identity among the three purified DT-diaphorases was finally confirmed by Ouchterlony immunodiffusion employing anti-serum raised against each enzyme preparation.

Promotion of tyrosinase folding in COS 7 cells by calnexin.

To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced approximately 2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When alpha-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immunoprecipitation of calnexin from extracts of [35S]methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER.