Glycosylation of P-glycoprotein in a multidrug-resistant KB cell line, and in the human tissues.

P-glycoprotein (P-gp) is thought to transport anti-cancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype. Immunohistochemistry reveals that P-gp is also expressed in normal human tissues, such as the adrenal gland, kidney, liver, and the capillary endothelium of the brain and testis. However, little is known about the structural and functional variations of P-gp in these tissues. With immunoblotting and photoaffinity labeling, we found that the molecular mass of P-gp in these tissues varied between 130-140 kDa. To clarify the post-translational modification of P-gp, we studied the biosynthesis of P-gp in a human multidrug-resistant cell line (KB-C2). We found that P-gp was produced in KB-C2 cells as a 125 kDa precursor and was slowly processed (t1/2 = 45-60 min) to the mature form of 140 kDa. In the presence of tunicamycin, a 120 kDa form of P-gp was synthesized and this form was no longer processed. Treating the 125 kDa precursor form with endo-beta-N-acetylglucosaminidase H (Endo H) and the 140 kDa mature form with N-glycanase diminished the molecular size of P-gp to that of the tunicamycin-treated form. N-Glycanase almost completely removed [3H]glucosamine labeling from P-gp. These data indicate that the major modification of P-gp is N-linked glycosylation. P-gps from KB-C2 cells, kidney and adrenal gland had a different lectin-binding capacity. There seems to be a variety of N-linked glycosylations in tissue and tumor P-gps.     

Characterization of cold-sensitive secY mutants of Escherichia coli.

Mutations which cause poor growth at a low temperature, which affect aspects of protein secretion, and which map in or around secY (prlA) were characterized. The prlA1012 mutant, previously shown to suppress a secA mutation, proved to have a wild-type secY gene, indicating that this mutation cannot be taken as genetic evidence for the secA-secY interaction. Two cold-sensitive mutants, the secY39 and secY40 mutants, which had been selected by their ability to enhance secA expression, contained single-amino-acid alterations in the same cytoplasmic domain of the SecY protein. Protein export in vivo was partially slowed down by the secY39 mutation at 37 to 39 degrees C, and the retardation was immediately and strikingly enhanced upon exposure to nonpermissive temperatures (15 to 23 degrees C). The rate of posttranslational translocation of the precursor to the OmpA protein (pro-OmpA protein) into wild-type membrane vesicles in vitro was only slightly affected by reaction temperatures ranging from 37 to 15 degrees C, and about 65% of OmpA was eventually sequestered at both temperatures. Membrane vesicles from the secY39 mutant were much less active in supporting pro-OmpA translocation even at 37 degrees C, at which about 20% sequestration was attained. At 15 degrees C, the activity of the mutant membrane decreased further. The rapid temperature response in vivo and the impaired in vitro translocation activity at low temperatures with the secY39 mutant support the notion that SecY, a membrane-embedded secretion factor, participates in protein translocation across the bacterial cytoplasmic membrane.     

Parathyroid hormone-related protein is a possible autocrine growth inhibitor for lymphocytes

Adult T-cell leukemia (ATL)-related cells have the ability to produce a newly-isolated calcium-regulating protein, parathyroid hormone-related protein (PTHrP). The present study revealed that lectin-stimulated normal lymphocytes produce immunoreactive (IR)-PTHrP. When the T-cell-enriched fraction was purified from normal lymphocytes and then treated with lectin, a similar amount of IR-PTHrP was detected, suggesting that IR-PTHrP is an actual product of T-lymphocytes. A biologically active fragment of PTHrP, PTHrP(1-34), suppressed DNA synthesis in lectin-stimulated lymphocytes at concentrations greater than 50 pg/mL; the same concentration range of IR-PTHrP detected in the cultured media of lectin-stimulated lymphocytes. Therefore, it is reasonable to postulate that PTHrP is a cytokine inhibiting the cellular growth of normal lymphocytes.     

Amphotericin B resistance is recessive in Chinese hamster hybrid cells

Previous studies from our laboratory have shown that Chinese hamster V79 cells mutated to high level resistance to amphotericin B have a lower cellular level of cholesterol, the target molecule for the polyene antibiotic. Two amphotericin B-resistant (AMB^R) mutants were each hybridized to their parental amphotericin B-sensitive (AMB^S) V79 cells. All the hybrids derived from AMB^R/AMB^S fusions were as sensitive to polyene antibiotics (amphotericin B, filipin, and pimaricin) as AMB^S/AMB^S hybrids. The AMB^R/AMB^S hybrids were found to contain cholesterol per phospholipids that is comparable to those in AMB^S or AMB^S/AMB^S. The analysis of hybrids formed between mutant and wild-type cells thus indicated that resistance to amphotericin B is a recessive marker, and that the cellular level of cholesterol is compensated in the AMB^S/AMB^R hybrids. Hybrids of AMB^R and AMB^R cells were all resistant, so that the three AMB^R mutants all fell into a single complementation group.     

Structural specificity of synthetic peptide adjuvant for induction of experimental allergic encephalomyelitis

The peptide N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), which has adjuvant activities, and 17 of its derivatives and analogs were synthesized and assayed to elucidate the structure necessary for adjuvant activity in induction of experimental allergic encephalomyelitis (EAE) in guinea pigs. The results revealed the importance of the d configuration and the α-carboxamide group of the isoglutaminyl residue of MDP for adjuvant activity. Replacement of the l-alanyl residue of MDP by d-alanine, but not by l-serine or glycine, resulted in a marked decrease in the activity. The β-methyl glycoside of MDP was found to be more active than the α-methyl derivative. 6-O-Stearoyl-N-acetylmuramyl-l-alanyl-d-isoglutamme showed activity.     

High-performance liquid chromatographic determination of short-chain aliphatic aldehydes using 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole as a fluorescence reagent

High-performance liquid chromatographic determination of four short-chain aliphatic aldehydes using fluorescence detection was carried out with 4-(N,N-dimethylaminosulphonyl)-7-hydrazino-2,1,3-benzoxadiazole (DBD-H). DBD-H derivatives with three aliphatic aldehydes — formaldehyde, acetaldehyde and propionaldehyde — were synthesized and their fluorescence properties were examined. Relative fluorescence intensities of these compounds in acetonitrile were ca. ten-fold larger than those in aqueous acetonitrile. DBD-hydrazones could be separated by reversed-phase chromatography using aqueous acetonitrile as eluent and detection at 560 nm with excitation at 445 nm. Submicromolar levels of formaldehyde, acetaldehyde, propionaldehyde and butylaldehyde could be determined. The HPLC procedure using propionaldehyde as internal standard was applied to the measurement of acetaldehyde levels in normal human plasma before and 30 min after ingestion of ethanol.     

Altered reactivity of immunoglobulin produced by human-human hybridoma cells transfected by pSV2-neo gene

The HB4C5 and HF10B4 cell lines are human-human hybridomas producing human IgM monoclonal antibodies (MAbs) reactive to porcine carboxypeptidase A (CPase), but not to double stranded DNA (ds DNA). We obtained G418-resistant HB4C5 and HF10B4 cells by an introduction of pSV2-neo DNA. Almost all of the G418-resistant clones produced MAbs reactive to not only the CPase but the ds DNA. The results of the inhibition ELISA suggested that the cross-reactivity of the antibodies from G418-resistant clones to CPase and ds DNA was responsible for the alteration on their antigen specificity. HB4C5 and HF10B4 cells and their G418-resistant clones produced antibodies having glycosylated chain. The antibodies produced by tunicamycin-treated G418-resistant subclones of HB4C5 and HF10B4 lost the ability to bind to ds DNA, but retained the ability to bind to CPase. These results suggest that an introduction of pSV2-neo DNA into these hybridomas alters the specificities of their MAbs, and that the alteration to antigen binding specificities of their MAbs may be associated with glycosylation of the MAbs by these hybridomas.     

Chemical identification and functional analysis of apocarotenoids involved in the development of arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizae formed between more than 80% of land plants and arbuscular mycorrhizal (AM) fungi represent the most widespread symbiosis on the earth. AM fungi facilitate the uptake of soil nutrients, especially phosphate, by plants, and in return obtain carbohydrates from hosts. Apocarotenoids, oxidative cleavage products of carotenoids, have been found to play a critical role in the establishment of AM symbiosis. Strigolactones previously isolated as seed-germination stimulants for root parasitic weeds act as a chemical signal for AM fungi during presymbiotic stages. Stimulation of carotenoid metabolism, leading to massive accumulation of mycorradicin and cyclohexenone derivatives, occurs during root colonization by AM fungi. This review highlights research into the chemical identification of arbuscular mycorrhiza-related apocarotenoids and their role in the regulation and establishment of AM symbiosis conducted in the past 10 years.