Effects of Hypoxia on the Activity of the Dopaminergic Neuron System in the Rat Striatum as Studied by In Vivo Brain Microdialysis

The purpose of the present study is to clarify the effects of hypoxia on the activity of the dopaminergic neurons in the brain and its mechanism of action. For this purpose, the effects of hypoxia on the extracellular levels of 3,4-dihy-droxyphenylethylamine (dopamine) were examined in the rat Striatum using in vivo brain microdialysis in the presence or absence of pretreatment with either tetrodotoxin (a blocker of voltage-dependent sodium channels) or nomifensine (a blocker of dopamine reuptake). Exposure to various degrees of hypoxia (15, 10, and 8% O₂ in N₂) increased dopamine levels in striatal dialysates to 200, 400, and 1,100%, respectively, of the control value. On reoxygenation, dopamine levels in the dialysates rapidly returned to the control level. Reexposure to hypoxia increased the dopamine levels to the same extent as during the first exposure. After addition of tetrodotoxin (40 mUM) to the perfusion fluid or pretreatment with nomifensine (100 mg/kg, i.p.), exposure to hypoxia no longer increased the dopamine levels. These results suggest that although hypoxia induces an increase in the extracellular dopamine levels (hence, an apparent increase in the activity of the dopaminergic neurons), this increase is not the result of an increase in dopamine release itself, but rather the result of inhibition of the dopamine reuptake mechanism.     

Different stability of ligand-receptor complex formed with two endothelin receptor species, ETA and ETB.

There are at least two types of endothelin receptors, ETA and ETB, present in various tissues. We found that although biotinylated ET-1 could bind to both ETA and ETB receptors, the stability of the formed ligand-receptor complexes was different. When the preformed complexes of receptor (solubilized from canine brain and lung membranes) and biotinylated ET-1 were subjected to avidin agarose column chromatography, most of the ETA activity was recovered in the pass-through fraction and the remainder was recovered in the 0.5 M KSCN eluate as ligand-free forms. On the other hand, the ETB activity bound firmly to the avidin agarose column was eluted with 1.5 M KSCN. The detection of the complex of 125I-ET-1 and its receptor by SDS-PAGE run at a low temperature was only possible with the ETB fractions and the complex of 125I-ET-1 and ETA was unstable during the separation. These results suggest that the conformation of the ligand binding sites of canine ETA and ETB as well as the stability of their ligand-receptor complexes to SDS are significantly different. Similar observations were also obtained for human ETA and ETB receptors.     

The polyphosphate kinase gene of Escherichia coli. Isolation and sequence of the ppk gene and membrane location of the protein.

Polyphosphate kinase (PPK) catalyzes the reversible transfer of the terminal phosphate of ATP to form a long-chain polyphosphate (polyP) (Ahn, K., and Kornberg, A. (1990) J. Biol. Chem. 265, 11734-11739). The Escherichia coli gene (ppk) encoding PPK has been cloned, sequenced, and overexpressed (about 100-fold). The gene possesses an open reading frame for 687 amino acids (mass of 80,278 Da). PPK has been purified from overproducing cells after release from attachment to the cell outer membrane; the purified soluble PPK reassociate with cell membrane fractions. About 850 molecules of PPK are found in a wild type cell.     

Sigma (sigma) antagonist BMY 14802 prevents methamphetamine-induced sensitization.

We have demonstrated for the first time that the sigma antagonist BMY 14802 prevents the development of behavioral sensitization induced by repeated administration of methamphetamine. Rats received an intraperitoneal injection of 15 or 30 mg/kg BMY 14802 followed by 2 mg/kg methamphetamine 30 min later. Unlike dopamine antagonists, BMY 14802 did not induce major changes in the acute motor effects of 2 mg/kg methamphetamine. Repeated administration of methamphetamine induced progressive augmentation of stereotyped behaviors and resulted in behavioral sensitization. However, repeated administration of methamphetamine in combination with BMY 14802 at either dose produced no increase in the intensity of stereotypy when compared with the first treatment. After a 7-day abstinence period, a challenge test with methamphetamine alone revealed supersensitivity of methamphetamine-sensitized rats to subsequent methamphetamine, whereas rats pretreated with repeated methamphetamine in combination with BMY 14802 exhibited no difference in the intensity of stereotypy from rats pretreated with repeated saline. These results suggest that sigma receptors play a crucial role in the induction of methamphetamine-induced sensitization.     

L-dopa administration enhances exocytotic dopamine release in vivo in the rat striatum.

Peripheral administration of L-3,4-dihydroxyphenylalanine (L-DOPA) methylester increased extracellular levels of DOPA and dopamine (DA) in the rat striatum monitored by in vivo brain microdialysis. The increase in DA levels persisted after inhibition of DA reuptake by nomifensine. Administration of blockers of voltage-dependent Na+ (tetrodotoxin) or Ca2+ (NKY-722) channels through the dialysis membrane completely eliminated the increase in DA levels. These results demonstrate that the L-DOPA-induced DA release is exocytotic in nature and hence, derived from neurons in the striatum.     

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.     

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.     

Preparation of a fluorescent derivative of benzoylecgonine, and preliminary studies of its application to the analysis of urine

A sensitive high-performance liquid chromatographic method using 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone (Br-DMEQ) as a fluorescent labeling reagent is described for the determination of benzoylecgonine (BE) and ecgonine (EC). The Br-DMEQ derivatives of BE and EC were separated on a C₁₈ column and detected at 455 nm with excitation at 370 nm. The detection limits of the proposed method were 18.7 fmol for BE and 12.5 pmol for EC at a signal-to-noise ratio of 3. Relative standard deviations of five replicate measurements were 1.94% (10 pmol) and 2.98% (50 pmol) for BE and 6.3% (250 pmol) and 5.62% (1.25 pmol) for EC. This method was applied to the determination of BE in human urine. BE was extracted from urine by solvent extraction with chloroform—isopropyl alcohol (9:1, v/v) solution. Levels of 2.5 · 10⁻⁸ M BE in urine (25 pmol/ml) could be determined.     

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.     

Point mutation of the p53 gene resulting in splicing inhibition in small cell lung carcinoma

The p53 gene is functionally inactivated mostly by point mutations resulting in amino acid substitutions in a wide variety of human cancers. We found a novel mutation of the p53 gene in a small cell lung carcinoma cell line, Lu-143. One of the allelic p53 genes was lost accompanied by loss of heterozygosity for chromosome 17. In the remaining allelic p53 gene, there was a single-base substitution of G to T at position 1 within the splice donor site of intron 7, and the mutated intron was not spliced out during the mRNA maturation process. As a result of this mutation, larger sized p53 mRNA was expressed and no p53 specific protein was detected in this cell line. These results suggest that mutations causing splicing abnormalities are one of the molecular mechanisms for the p53 gene inactivation in human cancer.