Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein J/clusterin

Glycoprotein 330 (gp330) is a member of a family of endocytic receptors related to the low density lipoprotein receptor. gp330 has previously been shown to bind a number of ligands in common with its family member, the low density lipoprotein receptor-related protein (LRP). To identify ligands specific for gp330 and relevant to its localization on epithelia such as in the mammary gland, gp330-Sepharose affinity chromatography was performed. As a result, a 70-kDa protein was selected from human milk and identified by protein sequencing to be apolipoprotein J/clusterin (apoJ). Solid-phase binding assays confirmed that gp330 bound to apoJ with high affinity (Kd = 14.2 nM). Similarly, gp330 bound to apoJ transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. LRP, however, showed no binding to apoJ in either type of assay. The binding of gp330 to apoJ could be competitively inhibited with excess apoJ as well as with the gp330 ligands apolipoprotein E, lipoprotein lipase, and the receptor-associated protein, a 39-kDa protein that acts to antagonize binding of all known ligands for gp330 and LRP. Several cultured cell lines that express gp330 and ones that do not express the receptor were examined for their ability to bind and internalize 125I-apoJ. Only cells that expressed gp330 endocytosed and degraded radiolabeled apoJ. Furthermore, F9 cells treated with retinoic acid and dibutyryl cyclic AMP to increase expression levels of gp330 displayed an increased capacity to internalize and degrade apoJ. Cellular internalization and degradation of radiolabeled apoJ could be inhibited with unlabeled apoJ, receptor-associated protein, and gp330 antibodies. The results indicate that gp330 but not LRP can bind to apoJ in vitro and that gp330 expressed by cells can mediate apoJ endocytosis leading to lysosomal degradation.     

Median-nerve neuropathy associated with chronic anterior dislocation of the lunate

Ten patients who had median-nerve neuropathy in association with chronic anterior dislocation of the lunate were managed operatively and were followed for an average of five years (range, three to eight years). The average time from the injury to the initial evaluation was twenty-one months (range, six to sixty-five months). All ten patients had pain as well as sensory and motor dysfunction in the distribution on the median nerve. Nerve-conduction-velocity studies revealed a delay in distal motor and sensory latencies in all patients; the distal motor latency averaged 12.5 milliseconds (range 5.6 to 18.6 milliseconds), and the distal sensory latency averaged 12.4 milliseconds (range, 4.8 to 16.8 milliseconds). Three patients had had a failed carpal tunnel release and needed excision of the lunate for decompression of the median nerve. In the other seven patients, three distinctive sites of nerve compression were identified: the volar and dorsal edges of the lunate and the proximal edge of the transverse carpal ligament. Excision of the osseous protuberance (excision of the lunate in three patients and a proximal-row carpectomy in four), combined with a release of the transverse carpal ligament, resulted in relief of the symptoms, functional improvement, and sensory and motor recovery in the distribution of the median nerve.     

Effects of in utero cocaine exposure on the expression of mRNAS encoding the dopamine transporter and the D1, D2 and D5 dopamine receptor subtypes in fetal rhesus monkey

The effects of in utero cocaine exposure on the development of the mRNAs encoding the dopamine transporter (DAT) and the D1, D2 and D5 dopamine receptor subtypes were determined in fetal monkey brains at day 45 and day 60 of gestation. Pregnant monkeys were treated with cocaine 3 mg/kg or saline i.m., four times a day from day 18 of gestation until the pregnancy was terminated at day 45 or day 60. The fetal brains were dissected, and tissue RNA extracted and quantified using ribonuclease protection assay analysis. In day 45 fetal monkeys, dopamine D1 and D2 receptor subtype mRNAs and DAT mRNA were found in low quantities both in control and cocaine-treated subjects. In day 60 fetal monkeys, D1 receptor mRNA levels were highest in the frontal cortex/striatal area, and low to moderate quantities were found in diencephalic and mesencephalic fetal brain regions. Dopamine D2 receptor mRNA levels were highest in the frontal cortex/striatal area, diencephalon and the midbrain, moderate in the brainstem and low in the caudal temporal lobe and surrounding cortical areas. Dopamine D5 receptor mRNA was expressed in low quantities throughout the day 60 fetal monkey brain, whereas DAT mRNA was found in the midbrain only. In utero cocaine exposure caused a significant increase in dopamine D1, D2 and D5 receptor subtype mRNAs in the frontal cortex/striatal area of day 60 fetal monkeys. These results support the hypothesis that dopamine synthesis and release may be reduced in cocaine-treated fetuses, which results in dopamine receptor up-regulation.     

Flow cytometric assay of modulation of P-glycoprotein function in whole blood by the multidrug resistance inhibitor GG918

Galactose-1-phosphate uridyl transferase (GALT) deficiency causes classical galactosemia in humans. Mice deficient in this enzyme were created by gene targeting. GALT-deficient mice develop biochemical features similar to those seen in humans with GALT deficiency, but fail to develop the pattern of acute toxicity seen in newborns with classical galactosemia. This study suggests that alternative routes of galactose metabolism are important in the pathogenesis of galactosemia.     

Deconvolution: a novel signal processing approach for determining activation time from fractionated electrograms and detecting infarcted tissue

BACKGROUND: Two important signal processing applications in electrophysiology are activation mapping and characterization of the tissue substrate from which electrograms are recorded. We hypothesize that a novel signal-processing method that uses deconvolution is more accurate than amplitude, derivative, and manual activation time estimates. We further hypothesize that deconvolution quantifies changes in morphology that detect electrograms recorded from regions of myocardial infarction. METHODS AND RESULTS: To determine the accuracy of activation time estimation, 600 unipolar electrograms were calculated with a detailed computer model using various degrees of coupling heterogeneity to model infarction. Local activation time was defined as the time of peak inward sodium current in the modeled myocyte closest to the electrode. Deconvolution, minimum derivative, and maximum amplitude were calculated. Two experienced electrophysiologists blinded to the computer-determined activation times marked their estimates of activation time. F tests compared the variance of activation time estimation for each method. To evaluate the performance of deconvolution to detect infarction, 380 unipolar electrograms were recorded from 10 dogs with infarcts resulting from ligation of the left anterior descending coronary artery. The amplitude, duration, number of inflections, peak frequency, bandwidth, minimum derivative, and deconvolution were calculated. Metrics were compared by Mann-Whitney rank-sum tests, and receiver operating curves were plotted. CONCLUSIONS: Deconvolution estimated local activation time more accurately than the other metrics (P < .0001). Furthermore, the algorithm quantified changes in morphology (P < .0001) with superior performance, detecting electrograms recorded from regions of myocardial infarction. Thus, deconvolution, which incorporates a priori knowledge of electrogram morphology, shows promise to improve present clinical metrics.     

2-Amino-4-methylpyridine as a potent inhibitor of inducible NO synthase activity in vitro and in vivo

1. The ability of 2-amino-4-methylpyridine to inhibit the catalytic activity of the inducible NO synthase (NOS II) enzyme was characterized in vitro and in vivo. 2. In vitro, 2-amino-4-methylpyridine inhibited NOS II activity derived from mouse RAW 264.7 cells with an IC50 of 6 nM. Enzyme kinetic studies indicated that inhibition is competitive with respect to arginine. 2-Amino-4-methylpyridine was less potent on human recombinant NOS II (IC50 = 40 nM) and was still less potent on human recombinant NOS I and NOS III (IC50 = 100 nM). NG-monomethyl-L-arginine (L-NMMA), N6-iminoethyl-L-lysine (L-NIL) and aminoguanidine were much weaker inhibitors of murine NOS II than 2-amino-4-methylpyridine but, unlike 2-amino-4-methylpyridine, retained similar activity on human recombinant NOS II. L-NMMA inhibited all three NOS isoforms with similar potency (IC50S 3-7 microM). In contrast, compared to activity on human recombinant NOS III, L-NIL displayed 10 x selectivity for murine NOS II and 11 x selectivity for human recombinant NOS II while aminoguanidine displayed 7.3 x selectivity for murine NOS II and 3.7 x selectivity for human recombinant NOS II. 3. Mouse RAW 264.7 macrophages produced high levels of nitrite when cultured overnight in the presence of lipopolysaccharide (LPS) and interferon-gamma. Addition of 2-amino-4-methylpyridine at the same time as the LPS and IFN-gamma, dose-dependently reduced the levels of nitrite (IC50 = 1.5 microM) without affecting the induction of NOS II protein. Increasing the extracellular concentration of arginine decreased the potency of 2-amino-4-methylpyridine but at concentrations up to 10 microM, 2-amino-4-methylpyridine did not inhibit the uptake of [3H]-arginine into the cell. Addition of 2-amino-4-methylpyridine after the enzyme was induced also dose-dependently inhibited nitrite production. Together, these data suggest that 2-amino-4-methylpyridine reduces cellular production of NO by competitive inhibition of the catalytic activity of NOS II, in agreement with results obtained from in vitro enzyme kinetic studies. 4. When infused i.v. in conscious unrestrained rats, 2-amino-4-methylpyridine inhibited the rise in plasma nitrate produced in response to intraperitoneal injection of LPS (ID50 = 0.009 mg kg-1 min-1). Larger doses of 2-amino-4-methylpyridine were required to raise mean arterial pressure in untreated conscious rats (ED50 = 0.060 mg kg-1 min-1) indicating 6.9 x selectivity for NOS II over NOS III in vivo. Under the same conditions, L-NMMA was nonselective while L-NIL and aminoguanidine displayed 5.2 x and 8.6 x selectivity respectively. All of these compounds caused significant increases in mean arterial pressure at doses above the ID50 for inhibition of NOS II activity in vivo. 5. 2-Amino-4-methylpyridine also inhibited LPS-induced elevation in plasma nitrate after either subcutaneous (ID50 = 0.3 mg kg-1) or oral (ID50 = 20.8 mg kg-1) administration. 6. These data indicate that 2-amino-4-methylpyridine is a potent inhibitor of NOS II activity in vitro and in vivo with a similar degree of isozyme selectivity to that of L-NIL and aminoguanidine in rodents.     

The production of interleukin-1 alpha immunoreactivity by human oviductal cells in a coculture system

PURPOSE: The aim of this study was to determine the concentration of interleukin-1 alpha in human embryo culture medium with or without oviductal cell coculture and to correlate the interleukin-1 alpha levels with pregnancy. METHODS: Culture media from 32 in vitro fertilization and embryo transfer cycles were assayed for interleukin-1 alpha by immunoassay technique. Human embryos were cultured in Earles' balanced salt solution supplemented with 15% preovulatory serum (sEBSS) in 16 of these cycles, while embryos in the rest of the cycles were cocultured with human oviductal cells in sEBSS. RESULTS: Both sEBSS and spent sEBSS after embryo culture contained low or undetectable levels of interleukin-1 alpha in the pregnant and nonpregnant cycles. On the other hand, oviductal cells significantly increased the amount of interleukin-1 alpha immunoreactivity in the conventional culture medium or coculture medium (P < 0.001, Mann-Whitney rank sum test). The concentrations of interleukin-1 alpha in the spent sEBSS after oviductal cell culture and after coculture with human embryos were 1.5 +/- 1.0 and 1.3 +/- 0.9 pg/ml, respectively. There was no difference in the interleukin-1 alpha concentration between the pregnant and the nonpregnant coculture cycles. CONCLUSIONS: These data showed that human oviductal cells produced interleukin-1 alpha immunoreactivity in a coculture system. However, this production could not be used as a marker for successful embryo implantation.