17beta-Estradiol-induced enhancement of estrogen receptor biosynthesis via MAPK pathway in mouse skeletal muscle myoblasts.

The skeletal muscle is one of the important target tissues for the actions of estrogen via both nuclear and extranuclear (non-genomic) pathways. However, there is a paucity of information about the receptor (ER) involved. The aim of this study was thus to explore the ER expression in skeletal muscle, and the influence of estrogen on it, by using C2C12 myoblasts derived from mouse skeletal muscle. Significant expression of a approximately 66-kD protein immunoreactive to ER type alpha (ERalpha) monoclonal antibody, which was comparable to that in ovary, was detected in the whole-cell (total) and nucleus-free (nonnuclear) fractions of C2C12 myoblasts. The expression level of these ER proteins increased in several hours with treatment with 17beta-estradiol (E2), which was preceded by the elevation of the ER mRNA level. This increase appeared to reflect the acceleration of de novo synthesis of ER protein, as proved by the (35)S-methionine immunoprecipitation method. A similar extent of fast increase in ER expression was also induced by a membrane-impermeable, BSA-conjugated estradiol (E2-BSA). Unexpectedly, the E2-induced increases in total and nonnuclear ER were further enhanced by the classic ER antagonists tamoxifen and ICI182,780 in a wide concentration range, implying some structural difference of the involved ER from the classical one. Treatment with the ERK1/2 inhibitor, PD98059 (10 microM), or the p38 MAPK-specific inhibitor, SB203580 (10 microM), greatly inhibited the E2-induced ER increase, while the protein kinase C (PKC) activator TPA (1 microM) enhanced it. These results collectively suggest that C2C12 skeletal myoblasts express a high level of ER, a considerable part of which is extranuclear. Further, the expression of ER in these cells may be significantly upregulated by estrogen itself via increased biosynthesis linked to membrane-bound ER and downstream MAPK-mediated signaling pathways.

Death receptor 3 (DR3) gene duplication in a chromosome region 1p36.3: gene duplication is more prevalent in rheumatoid arthritis.

The death receptor 3 (DR3) gene is a member of the apoptosis-inducing Fas gene family. In the current study, fluorescence in situ hybridization (FISH) and Fiber-FISH revealed the existence of a second DR3 gene approximately 200 kb upstream of the original DR3 gene. The existence of the duplicated DR3 gene was confirmed by sequencing the corresponding human artificial chromosome clones as well as with quantitative PCR that measured the ratio of the DR3 gene mutation (Rm), intrinsic to rheumatoid arthritis (RA) patients, by simultaneous amplification of the normal and mutated DR3 sequences. The DR3 gene duplication measured by FISH was found to be more frequent in patients with RA as compared to healthy individuals. We therefore surmise that the human DR3 gene can be duplicated and that this gene duplication is more prevalent in patients with RA.

Steroid and xenobiotic receptor (SXR) is a key system for the acquisition of cisplatin resistance in endometrial cancer cells.

A novel steroid and xenobiotic receptor (SXR) may be involved in chemoresistance, and we studied this receptor in endometrial cancer cell lines. The cisplatin (CDDP)-sensitive Ishikawa cell line and its CDDP-resistant sub-clone (ISIW+) were used. ISIW+ cells showed a much higher SXR expression. When Ishikawa cells were cultured with SXR anti-sense oligonucleotides (AS), the cells failed to pass crisis and did not gain cisplatin resistance. In an experiment using SCID mice, all AS-treated animals survived, whereas controls had 50% survival at 35 days. The present data indicate that SXR is a key system to induce, maintain and reverse a cisplatin-resistant phenotype in endometrial cancer cells.

Agranulocytosis possibly caused by ranitidine in a patient with renal failure.

A 70-year-old Japanese woman with renal dysfunction under hemodialysis presented with vomiting and chill with fever. Over the previous 24 weeks she had been taking 75 mg of ranitidine after hemodialysis. Other medications taken were prednisolone, furosemide, alpha-calcidol, amlodipine and calcium carbonate. Before starting ranitidine, she had been treated with famotidine for about 2 years without complication. Hematological inspection on admission revealed agranulocytosis with WBC of 400/mm3. Ranitidine was discontinued and granulocyte colony-stimulating factor (G-CSF) was started. On Day 3, laboratory data showed slight improvement of cytopenia with WBC of 1,000/mm3. On Day 6, her hemogram showed marked improvement with WBC of 11,700/mm3 and G-CSF was discontinued. She was discharged on Day 10. Several cases describing ranitidine-induced cytopenia are associated with the use of ranitidine at a dose of 150 mg/day or higher, and adverse reactions were found within 2-35 days after beginning ranitidine treatment. In the case described here, however, the adverse reaction occurred after a longer treatment period with ranitidine at a lower dose. In conclusion, ranitidine should be administered with great caution to patients with severe renal dysfunction.

De novo-designed peptide transforms Golgi-specific lipids into Golgi-like nanotubules.

Cellular organelles, such as the Golgi apparatus and the endoplasmic reticulum, adopt characteristic structures depending on their function. While the tubular shapes of these structures result from complex protein-lipid interactions that are not fully understood, some fundamental machinery must be required. We show here that a de novo-designed 18-mer amphipathic alpha-helical peptide, Hel 13-5, transforms spherical liposomes made from a Golgi-specific phospholipid mixture into nanotubules on the scale of and resembling the shape of the nanotubules that form the Golgi apparatus. Furthermore, we show that that the size and the shape of such nanotubules depend on lipid composition and peptide properties such as length and the ratio of hydrophobic to hydrophilic amino acids. Although the question of precisely how nature engineers organellar membranes remains unknown, our simple novel system provides a basic set of tools to begin addressing this question.

Intracellular cleavage of glycosylphosphatidylinositol by phospholipase D induces activation of protein kinase Calpha.

Many proteins are anchored to the cell membrane by glycosylphosphatidylinositol (GPI). One of the functions proposed for the GPI anchor is as a possible mediator in signal transduction through its hydrolysis. GPI-specific phospholipase D (GPI-PLD) is a secretory protein that is suggested to be involved in the release of GPI-anchored protein from the membrane. In the present study we examined how GPI-PLD is involved in signal transduction. When introduced exogenously and overexpressed in cells, GPI-PLD cleaved the GPI anchors in the early secretory pathway, possibly in the endoplasmic reticulum, resulting in an increased production of diacylglycerol. Experiments in vitro and in vivo showed that the association of protein kinase Calpha (PKCalpha) with membranes was increased markedly by expression of GPI-PLD in cells. Furthermore, sucrose-density-gradient centrifugation and immunofluorescence microscopy demonstrated that PKCalpha was translocated to the endoplasmic reticulum membrane in cells expressing GPI-PLD, in contrast with its association with the plasma membrane in cells treated with PMA. We also confirmed that the phosphorylation of c-Fos as well as PKCalpha itself was greatly enhanced by the expression of GPI-PLD. Taken together, these results suggest that GPI-PLD is involved in intracellular cleavage of the GPI anchor, which is a new potential source of diacylglycerol production to activate PKCalpha.

New Prognostic Factors Associated with Long-term Survival in Node-Negative Breast Cancer Patients.

BACKGROUND: This study was undertaken to determine the absolute and relative value of angiogenesis, proliferating cell nuclear antigen (PCNA) and conventional prognostic factors in predicting relapse-free survival (RFS) and overall survival (OS) rates associated with long-term survival in Japanese patients with node-negative breast cancer. PATIENTS AND METHODS: Two hundred patients with histological node-negative breast cancer were studied. We investigated nine clinicopathological factors, including angiogenesis, PCNA using per-manent-section immunohistochemistry, clinicaltumor size, histological grade (HG), tumor necrosis, lymphatic vessel invasion (LVI), histological extension, histological classification, and infiltrating growth (INF), followed for a median of 10 years (range, 1 to 20). RESULTS: Twenty-one patients (10.5%) had recurrence and 15 patients (7.5%) died of breast cancer. Univariate analysis showed that PCNA, clinical tumor size, HG, angiogenesis, and LVI were significantly predictive of 20-year RFS or OS. Tumor necrosis was significantly predictive of OS, not of RFS. Multi-variate analysis showed that clinical tumor size (P = 0.0003), angiogenesis (P = 0.0003), PCNA (P = 0.0064), and HG (P = 0.0401) were significant independent prognostic factors for RFS. PCNA (P< 0.0001) and clinical tumor size (P = 0.0112) were significant independent prognostic factors for OS, while angiogenesis was a borderline significant factor. CONCLUSION: PCNA and angiogenesis were important new prognostic factors in node-negative breast cancer patients.

Posttranslational modification of glycosylphosphatidylinositol (GPI)-specific phospholipase D and its activity in cleavage of GPI anchors.

Human glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was exogenously expressed in mammalian CHO cells and in insect H5 cells. GPI-PLD was initially synthesized as a 105-kDa form and then secreted as a mature 115-kDa form from the CHO cells, whereas it was secreted as an immature 98-kDa form from the H5 cells. The difference of the molecular forms was caused by its oligosaccharide processing in the two cell lines. These forms showed a different reactivity to anti-C-terminal peptide of GPI-PLD; the 105-kDa and 98-kDa forms were directly recognized by the antibodies, whereas the 115-kDa form was immunoreactive only after being denatured. In an in vitro assay, the 98-kDa form but not the 115-kDa form was able to release a significant amount of GPI-anchored proteins from intact membranes, although the two forms had the same GPI-anchor cleavage activity in the presence of detergents. In addition, a GPI-anchored protein, when coexpressed in CHO cells, was intracellularly cleaved by GPI-PLD in the secretory pathway. Taken together, these results suggest that GPI-PLD undergoes a conformational change by posttranslational modification, which affects its immunoreactive and enzymatic properties.

Decision analysis for treatment of early stage prostate cancer.

We performed a decision analysis to evaluate the usefulness of pretreatment prediction of clinically significant or insignificant tumor in patients with prostate-specific antigen (PSA)-detected stage T1c prostate cancer nonpalpable on rectal examination. Analysis was done for otherwise healthy subjects with 20 years of life expectancy. The prevalence of insignificant tumor among those with T1c prostate cancer was initially assumed to be 0.2. Quality-adjusted life expectancy was calculated and compared between 2 strategies; one with prediction-based selection of either radical prostatectomy or watchful waiting and the other with unselective assignment of one of the treatments. The selection strategy was superior when the sensitivity and specificity for detecting clinically significant tumor were 0.92 and 0.73, respectively, as reported by Epstein et al. (1994) using criteria of PSA density and Gleason score in a needle biopsy specimen. Sensitivity analysis revealed that the prediction-based selection strategy is preferred, with sensitivity and specificity constant, when the prevalence of insignificant tumor exceeds 0.16. On the other hand, when the prevalence of insignificant tumor is kept constant at 0.2, sensitivity should be 0.85 or higher for the prediction strategy to be preferred. As the prevalence of insignificant tumor among those with T1c prostate cancer increased, the prediction-based selection strategy is preferred with lower values of sensitivity and specificity for detecting significant tumor. These results suggest that a selective treatment strategy of either radical or conservative treatment based on pretreatment prediction for significant tumor is a beneficial alternative to radical prostatectomy unselectively assigned to all patients at the T1c stage, if a reasonable accuracy in prediction is attained.

Phosphorylation of the vesicle docking protein p115 regulates its association with the Golgi membrane.

The vesicle docking protein p115 was found to be phosphorylated in a cell cycle-specific manner; it was found phosphorylated in interphase but not in mitotic cells. During interphase, however, two forms of p115 were detected in the cells; the phosphorylated form was found exclusively in cytosol, whereas the unphosphorylated form was associated with membranes, mostly of the Golgi complex. The latter form was released from the membranes upon phosphorylation. Mutational analysis revealed that the phosphorylation site of p115 was the Ser942 residue in the C-terminal acidic domain. A mutant with a single substitution of Ser942 --> Ala markedly increased its association with the Golgi membrane. Another mutant with Ser942 --> Asp was able to associate with the membrane, although at a decreased level, indicating that the dissociation of p115 from the membrane is not simply due to the negative charge of phosphorylated Ser942. Taken together, these results suggest that the phosphorylation of Ser942 at the C-terminal acidic domain regulates the interaction of p115 with the Golgi membrane, possibly taking part in the regulatory mechanism of vesicular transport.

Nordihydroguaiaretic acid blocks protein transport in the secretory pathway causing redistribution of Golgi proteins into the endoplasmic reticulum.

We have investigated the effect of nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, on the intracellular protein transport and the structure of the Golgi complex. Pulse-chase experiments and immunoelectron microscopy showed that NDGA strongly inhibits the transport of newly synthesized secretory proteins to the Golgi complex resulting in their accumulation in the endoplasmic reticulum (ER). Despite their retention in the ER, oligosaccharides of secretory and ER-resident proteins were processed to endoglycosidase H-resistant forms, raising the possibility that oligosaccharide-processing enzymes are redistributed from the Golgi to the ER. Morphological observations further revealed that alpha-mannosidase II (a cis/medial-Golgi marker), but not TGN38 (a trans-Golgi network marker), rapidly redistributes to the ER in the presence of NDGA, resulting in the disappearance of the characteristic Golgi structure. Upon removal of the drug, the Golgi complex was reassembled into the normal structure as judged by perinuclear staining of alpha-mannosidase II and by restoration of the secretion. These effects of NDGA are quite similar to those of brefeldin A. However, unlike brefeldin A, NDGA did not cause a dissociation of beta-coatomer protein, a subunit of coatomer, from the Golgi membrane. On the contrary, NDGA exerted the stabilizing effect on beta-coatomer protein/membrane interaction against the dissociation caused by brefeldin A and ATP depletion. Taken together, these results indicate that NDGA is a potent agent disrupting the structure and function of the Golgi complex with a mechanism different from those known for other drugs reported so far.

An alpha2-macroglobulin-serine proteinase complex from human carcinomatous ascites and pleural effusion: isolation, monoclonal antibody preparation, and immunohistochemical study.

A protein with the apparent molecular mass of 720 kDa which hydrolyzes anilide substrates of p-guanidino-L-phenylalanine was purified from ascites and pleural effusion of patients with pulmonary, breast, gastric, and ovarian cancers by chromatographic techniques. When this protein was separated on SDS-PAGE on nonreducing conditions, two bands corresponding to 720 and 360 kDa were seen to have gelatin-digestive activity in zymography assay. Moreover, when it separated by SDS-PAGE on reducing conditions, it migrated as several bands up to 180 kDa. The N-terminal amino acid sequence and immunoreactivity of anti-alpha2-macroglobulin polyclonal antibody revealed that the 180-kDa band was intact alpha2-macroglobulin. The hydrolytic activity of this complex was completely inhibited by diisopropyl fluorophosphate (DFP) and p-amidinophenylmethanesulfonyl fluoride. In addition, the 65-kDa protein observed under reducing conditions bound 3H-labeled DFP. These results suggest that the purified protein is a complex of the plasma proteinase inhibitor alpha2-macroglobulin and a serine proteinase. Several monoclonal antibodies were obtained when the purified complex was used as an antigen. One of these antibodies, which was immunoreactive to this complex but not to alpha2-macroglobulin, gave a positive band corresponding to 65 kDa on SDS-PAGE under reducing conditions. Use of this antibody in immunohistochemical studies revealed immunoreactivities in numerous neoplastic tissues with strong activity in advanced gastric cancers (e.g., poorly differentiated adenocarcinoma). In addition, strong cross-reactivity was detected in glandular cells of the fetus intestine.

Ca2+-dependent interaction of the growth-associated protein GAP-43 with the synaptic core complex.

The synaptic vesicle exocytosis occurs by a highly regulated mechanism: syntaxin and 25 kDa synaptosome-associated protein (SNAP-25) are assembled with vesicle-associated membrane protein (VAMP) to form a synaptic core complex and then synaptotagmin participates as a Ca2+ sensor in the final step of membrane fusion. The 43 kDa growth-associated protein GAP-43 is a nerve-specific protein that is predominantly localized in the axonal growth cones and presynaptic terminal membrane. In the present study we have examined a possible interaction of GAP-43 with components involved in the exocytosis. GAP-43 was found to interact with syntaxin, SNAP-25 and VAMP in rat brain tissues and nerve growth factor-dependently differentiated PC12 cells, but not in undifferentiated PC12 cells. GAP-43 also interacted with synaptotagmin and calmodulin. These interactions of GAP-43 could be detected only when chemical cross-linking of proteins was performed before they were solubilized from the membranes with detergents, in contrast with the interaction of the synaptic core complex, which was detected without cross-linking. Experiments in vitro showed that the interaction of GAP-43 with these proteins occurred Ca2+-dependently; its maximum binding with the core complex was observed at 100 microM Ca2+, whereas that of syntaxin with synaptotagmin was at 200 microM Ca2+. These values of Ca2+ concentration are close to that required for the Ca2+-dependent release of neurotransmitters. Furthermore we observed that the interaction in vitro of GAP-43 with the synaptic core complex was coupled with protein kinase C-mediated phosphorylation of GAP-43. Taken together, our results suggest a novel function of GAP-43 that is involved in the Ca2+-dependent fusion of synaptic vesicles.

Involvement of intracellular cyclic GMP and cyclic GMP-dependent protein kinase in alpha-elastin-induced macrophage chemotaxis.

alpha-Elastin with an average molecular mass of 70 kDa, an oxalic acid fragmentation product of highly purified insoluble elastin, induced the migration of macrophages, with maximum activity at 10(-1) microg/ml. Relative to the positive control of 10(-8) M N-formylmethionyl-leucyl-phenylalanine (fMLP), the responsiveness of macrophages to alpha-elastin was nearly the same. Checkerboard analysis demonstrated that the cell movement is chemotaxis and not chemokinesis. A homologous deactivation test showed the possibility of the existence of alpha-elastin-recognizing sites on macrophages. In connection with macrophage chemotaxis in response to alpha-elastin, the intracellular signaling pathway was examined. The guanosine 3', 5'-cyclic monophosphate (cGMP) level was enhanced in macrophages stimulated by alpha-elastin, whereas the adenosine 3',5'-cyclic monophosphate (cAMP) level was not. Chemotaxis assaying of macrophages treated with 8-Br cGMP- and dibutyryl cAMP-loaded macrophages indicated that cGMP promotes cell movement and cAMP suppresses cell locomotion. The possible involvement of protein kinases in the alpha-elastin signaling pathway was explored by use of inhibitors specific for cGMP-dependent protein kinase (PKG), cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and tyrosine kinase. The macrophage chemotactic response to alpha-elastin was inhibited by the PKG inhibitor, but not by the PKA, PKC, or tyrosine kinase inhibitor. These results suggested that the increase in the cGMP level and the activation of PKG in macrophages are involved in alpha-elastin induced macrophage chemotaxis.

Mainz pouch with appendix-umbilical stoma using catheterizable conduit elongated with continuous cecal segment: a case report.

The Mainz pouch with appendix-umbilical stoma is a very stable method for continent, self-catheterizable urinary reservoir in the presence of a healthy appendix. If the appendix is too short or an unexpected stenosis is seen at its distal portion, the elongation of the conduit using a part of the cecum and the implantation of the conduit to the pouch by the Mitrofanoff method can be a good alternative procedure. We herein report our experience in a 53-year-old male with high grade, invasive bladder tumor, who underwent cystourethrectomy and appendix Mainz pouch operation using the above technique.

Bip/GRP78 but not calnexin associates with a precursor of glycosylphosphatidylinositol-anchored protein.

When fused in-frame with a C-terminal propeptide of placental alkaline phosphatase (PLAP), rat alpha 2u-globulin (alpha GL), a nonglycosylated secretory protein, was expressed on the cell surface as a glycosylphosphatidylinositol (GPI)-linked chimaeric protein (alpha GL-PLAP). In contrast with the wild-type alpha GL-PLAP, a mutant, in which Asp at the cleavage/attachment site of GPI was replaced by Trp, failed to become a GPI-linked mature form and was retained as a precursor form within the cell [Oda, Cheng, Saku, Takami, Sohda, Misumi, Ikehara and Millán (1994) Biochem. J. 301, 577-583]. To elucidate the molecular interactions involved in the retention of the proform within the cell, we examined the association of the proform with molecular chaperones in the endoplasmic reticulum (ER). Antibody against the ER retrieval motif KDEL coimmunoprecipitated a 25 kDa proform, but not a 22 kDa GPI-linked mature form. Pulse-chase experiments showed that the wild-type alpha GL-PLAP with a cleavable propeptide was converted into the mature form, whereas the mutant alpha GL-PLAP with an uncleavable propeptide remained associated with ER-resident proteins with a KDEL motif and underwent rapid degradation in a pre-Golgi compartment. Chemical cross-linking studies showed that, of the several ER-resident proteins immunoreactive with the anti-KDEL antibody, a 78 kDa protein was the only protein associated with the proform. Furthermore this 78 kDa protein was dissociated from the precursor molecule on incubation with ATP, allowing us tentatively to assign it as Bip/GRP78. Anticalnexin antibody, however, failed to coprecipitate any form of the chimaeric protein. Immunoelectron microscopy showed that the proform with the uncleavable propeptide was localized in the ER, but not detected in the Golgi apparatus or plasma membranes. Taken together, these results suggest that Bip/GRP78 is associated with pro alpha GL-PLAP and retains it within the ER until pro alpha GL-PLAP is either modified by GPI or degraded, thereby participating in the quality control of this GPI-linked chimaeric protein.

Two novel point mutations in the lecithin:cholesterol acyltransferase (LCAT) gene resulting in LCAT deficiency: LCAT (G873 deletion) and LCAT (Gly344-->Ser).

We investigated the genetic defects in two patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. Their clinical manifestations including corneal opacities, anemia, proteinuria, and hypoalphalipoproteinemia were identical for familial LCAT deficiency. Their LCAT activities and the cholesterol esterification rate (CER) were nearly zero, and their LCAT masses were below 10% of normal control values. Sequence analysis of the amplified DNA of case 1 revealed one base deletion of G at base 873 (first position of Val264) in exon 6, leading to a premature termination by frameshift. Sequence analysis of amplified DNA of case 2 revealed a single G to A converting Gly (GGT) to Ser (AGT) substitution at residue 344. When COS-1 cells were transfected with these mutants, LCAT activity in the medium was nearly zero, and the LCAT mass was undetectable (< 0.01 microgram/ml). In contrast, LCAT activity in the medium of COS-1 cells, transfected with wild-type LCAT, was 1.7 nmol/h per ml and the LCAT mass was 0.09 micrograms/ml. The LCAT mass in the cell lysates of the mutants was less than 12% of control for case 1 and 18% of control for case 2. Northern blot analysis of the mRNA of COS-1 cells transfected with the mutants showed the same amounts of LCAT mRNA as compared with wild-type LCAT. Biosynthesis of mutant LCATs was analyzed by pulse-chase and immunocytochemistry in transfected baby hamster kidney cells. SDS-PAGE/fluorography demonstrated that wild-type LCAT was synthesized as a high-mannose type of 56 kDa, which was very slowly converted to a mature form of 67 kDa and was secreted into the media. In contrast to the wild-type LCAT, the mutant precursors were not processed into the mature form but slowly degraded along with chase times. On steady and continuous labeling in the case of wild-type LCAT, the mature 67 kDa form was observed in both the cell lysate and media, whereas no mature form was detected in the cell lysates and media which were transfected mutant LCATs. These data suggest that the mutant LCATs are actually synthesized in an amount comparable to that of wild-type, but they are slowly degraded without being processed into the mature form. The immunocytochemistry revealed that mutant LCATs were mainly retained in the endoplasmic reticulum. These data suggest that these two mutations may disrupt the mutant LCATs' transport from the endoplasmic reticulum into Golgi apparatus, resulting in LCAT deficiency.

Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase.

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.

Triazolam and nitrazepam use in elderly outpatients.

OBJECTIVE: To determine adverse reactions and effects on sleep among three groups of patients: those taking triazolam, those taking nitrazepam, and a control group. DESIGN: Telephone interviews. PATIENTS: Forty-seven patients taking triazolam, 36 taking nitrazepam, and 40 control patients. All study participants were outpatients over 60 years of age. RESULTS: The rate of awakening in the middle of sleep was not significantly different among patients taking triazolam (61.7 percent) and those taking nitrazepam (69.4 percent). Incidence of nocturia, the primary reason for awakening, was not significantly different between triazolam- (36.2 percent) and nitrazepam-taking patients (41.7 percent). The rate of having difficulty falling back to sleep was significantly different among triazolam (62.1 percent) and nitrazepam (8 percent), and triazolam and control (11.1 percent) groups (p < 0.01). No difference was evident, however, between nitrazepam and control groups. CONCLUSIONS: Patients taking nitrazepam have less difficulty returning to sleep compared with those who take triazolam. Thus, for elderly patients who awaken because of nocturia, nitrazepam may be more appropriate therapy.

An active-site mutation (Gly633-->Arg) of dipeptidyl peptidase IV causes its retention and rapid degradation in the endoplasmic reticulum.

Dipeptidyl peptidase IV (DPPIV), a serine protease expressed on the cell surface, is deficient in a Fischer rat substrain. Northern blot analysis showed no difference in the size and amount of DPPIV mRNA between normal (344/NC) and deficient (344/CRJ) rats. Cloning and sequencing of DPPIV cDNAs revealed a G to A transition at nucleotide 1897 in the cDNA sequence of 344/CRJ, which leads to substitution of Gly633-->Arg in the active-site sequence Gly629-Trp-Ser-Tyr-Gly633 determined for the wild-type DPPIV [Ogata, S., Misumi, Y., Takami, N., Oda, K., & Ikehara, Y. (1992) Biochemistry 31, 2582-2587]. Pulse-chase experiments with hepatocytes showed that the wild-type DPPIV was initially synthesized as a 103-kDa form with high-mannose-type oligosaccharides, which was processed to a mature form of 109 kDa with the complex type during intracellular transport. In contrast, the mutant DPPIV, although being synthesized as the 103-kDa form, was rapidly degraded without being processed to the mature form. Site-directed mutagenesis of the wild-type and mutant cDNAs and their transfection/expression in COS-1 cells confirmed that the single substitution of Gly633-->Arg is sufficient to cause the rapid intracellular degradation of DPPIV. Immunoelectron-microscopic observations showed that the mutant DPPIV was detectable only in the endoplasmic reticulum (ER), in contrast to the distribution of the wild-type DPPIV in the Golgi complex and on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)