Experience with Sephadex gel filtration in assessing the risk of bilirubin encephalopathy in neonatal jaundice

Clinical experience with Sephadex gel filtration for evaluation of the bilirubin binding affinity of serum in neonatal jaundice is reported. In serum specimens from 362 jaundiced neonates the results of the test were in most cases in good agreement with the independently determined decision to perform exchange transfusion. In all relevant cases of the 24 with bilirubin encephalopathy the Sephadex test was strongly positive. The test was positive at lower bilirubin levels and bilirubin:albumin molar ratios in preterm as compared with term neonates, especially those in poor clinical conditions. Among term infants the test indicated an increase in bilirubin binding affinity after the fifth day of life. The Sephadex test appears to be of practical value in supplementing and refining existing criteria for exchange transfusion.     

Protection against preterm delivery in mice by urinary trypsin inhibitor

OBJECTIVE: To investigate the precise mechanism by which urinary trypsin inhibitor suppresses cytokine production in the prevention of preterm delivery. METHODS: In vivo and in vitro studies were performed using ascites and peritoneal macrophages obtained on day 15 of pregnancy from female C3H/HeN mice that had been impregnated by B6D2F1 male mice. Lipopolysaccharide receptor, the intracellular signal transduction system, and nuclear factor-kappaB level were examined. RESULTS: In the in vivo study, we found that urinary trypsin inhibitor ameliorated the deterioration of intraperitoneal conditions induced by lipopolysaccharide (ie, increases in ascitic volume, peritoneal cell count, and tumor necrosis factor-alpha level) and caused a decrease in the binding of lipopolysaccharide to mouse macrophages. In the in vitro studies, urinary trypsin inhibitor decreased the binding capacity of lipopolysaccharide for its receptor, blocked the intracellular signal transduction induced by lipopolysaccharide, and decreased the nuclear factor-kappaB level. Increases were induced in the binding capacity of the macrophages for urinary trypsin inhibitor and its incorporation into them in the presence of lipopolysaccharide. CONCLUSION: We postulate that urinary trypsin inhibitor may suppress the production of inflammatory cytokines induced by lipopolysaccharide in mouse peritoneal macrophages through suppression of the lipopolysaccharide receptor, inhibition of the intracellular signal transduction system, and decrease in the nuclear factor-kappaB level.     

Polyethylene glycol-modified bilirubin oxidase improves hepatic energy charge and urinary prostaglandin levels in rats with obstructive jaundice

BACKGROUNDS/AIMS: No study has so far been conducted to clarify whether the presence of hyperbilirubinemia is detrimental to liver and renal functions. In the present study, the effects of polyethylene glycol-modified bilirubin oxidase (PEG-BOX) therapy on liver and renal function tests, hepatic energy charge and urinary prostaglandin levels were evaluated in a rat model of obstructive jaundice. METHODS: Sprague-Dawley rats were used in the experimental model of obstructive jaundice. PEG-BOX or an equivalent amount of PEG alone was intravenously injected into the animals and sampling of blood and urine, and liver harvesting were done sequentially after bile duct ligation. RESULTS: Conventional liver function tests showed no difference between PEG-BOX and control groups. However, bilirubin concentrations in the peripheral blood and liver tissue specimens markedly decreased, and the hepatic energy charge significantly increased in the PEG-BOX group as compared to controls. The blood concentration of bile acid was lower, but its urinary excretion was higher in the PEG-BOX group than in the control group. In vitro incubation of PEG-BOX with serum from rats with obstructive jaundice decreased the concentration of bilirubin but not that of bile acid. The urinary levels of prostaglandin E2 and the thromboxane B2/6-keto-prostaglandin Fla ratio were significantly lower in the PEG-BOX group than in the control group. CONCLUSIONS: The systemic reduction of bilirubin concentration may contribute to normalization of the urinary levels of prostaglandins and thromboxane B2, to decrease in serum bile acid levels, and to improvement of the hepatic energy charge in obstructive jaundice. These findings suggest that preoperative improvement of jaundice may be beneficial to patients with obstructive jaundice.     

C1 inhibitor and diagnosis of hereditary angioedema in newborns

Symptoms of hereditary angioedema may present during the child's first years. Attacks may be a particular threat to the narrower airway of the child. An early diagnosis is most valuable because effective C1 inhibitor (C1 INH) concentrate is available. We present a reference area for the antigenic and functional determination of C1 INH by using uncontaminated umbilical cord blood from 80 normal newborns collected by puncturing vessels in the newly delivered placenta. We examined two full-term babies (1 and 2) from mothers with hereditary angioedema type I the same way. The concentration of C1 INH antigen was determined by radial immunodiffusion. The C1 INH functional assay was based on the addition of a known quantity of C1s, which enzymatically splits a chromogenic substrate. The test was performed in the presence of methylamine and heparin in a kinetic microtiter plate assay. Citrated plasma was used in both assays. The data obtained in the 80 cord blood samples (2.5-97.5 percentile) were 0.11-0.22 g/L for C1 INH antigen (adults, 0.15-0.33 g/L) and 47.2-85.9% for C1 INH function (percentage of adults). In cord blood, baby 1 had an antigenic value of 0.12 g/L (7.5 percentile) and C1 INH function of 61.8% (42 percentile). The corresponding values for baby 2 in cord blood were less than 0.05 g/L (0.106 g/L < 2.5 percentile) and 34.3% (12.9% < 2.5 percentile). Baby 2 had markedly lower C4 values yet much higher C4 activation products than baby 1. At 4 mo, baby 1 had an antigenic C1 INH value of 0.24 g/L.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic studies of the binding of bilirubin by ligandin and aminoazo-dye-binding protein A

Ligandin and aminoazo-dye-binding protein A both bind bilirubin at a single site. Quantitative studies of the interactions using difference spectrophotometry show that at pH 7.0, protein A binds the tetrapyrrole with an association constant (K) greater than or equal to 2 X 10(7) litre/mol, whereas binding by ligandin is slightly weaker (K = 7 X 10(6) litre/mol) at this pH. The protein-bilirubin complexes give rise to absorption and fluorescence spectra quite different from those of unbound bilirubin and also to large Cotton effects. It appears that on binding to both proteins, the ligand is forced into a rigid twisted configuration in a hydrophobic environment. Ligandin and protein A resemble serum albumin in their interactions with bilirubin.     

Language discrimination by newborns: Toward an understanding of the role of rhythm.

Three experiments investigated the ability of French newborns to discriminate between sets of sentences in different foreign languages. The sentences were low-pass filtered to reduce segmental information while sparing prosodic information. Infants discriminated between stress-timed English and mora-timed Japanese (Experiment 1) but failed to discriminate between stress-timed English and stress-timed Dutch (Experiment 2). In Experiment 3, infants heard different combinations of sentences from English, Dutch, Spanish, and Italian. Discrimination was observed only when English and Dutch sentences were contrasted with Spanish and Italian sentences. These results suggest that newborns use prosodic and, more specifically, rhythmic information to classify utterances into broad language classes defined according to global rhythmic properties. Implications of this for the acquisition of the rhythmic properties of the native language are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Altered biliary bilirubin profile in patients with persistent hyperbilirubinaemia after hepatic resection: analysis of bile bilirubin subfractions by high-performance liquid chromatography

PURPOSE: Although vagus nerve stimulation (VNS) is now marketed throughout most of the world as a treatment for drug-resistant epilepsy, the therapeutic mechanism of action of VNS-induced seizure suppression has not yet been established. Elucidation of this mechanism is an important first step in the development of strategies to improve VNS efficacy. Because the locus coeruleus (LC) has been implicated in the antinociceptive effects of VNS, we chemically lesioned the LC in the present study to determine if it is a critical structure involved in the anticonvulsant mechanisms of VNS. METHODS: Rats were chronically depleted of norepinephrine (NE) by a bilateral infusion of 6-hydroxydopamine (6-OHDA) into the LC. Two weeks later, they were tested with maximal electroshock (MES) to assess VNS-induced seizure suppression. In another experiment, the LC was acutely inactivated with lidocaine, and seizure suppression was tested in a similar fashion. RESULTS: VNS significantly reduced seizure severities of control rats. However, in animals with chronic or acute LC lesions, VNS-induced seizure suppression was attenuated. CONCLUSIONS: Our data indicate that the LC is involved in the circuitry necessary for the anticonvulsant effects of VNS. Seizure suppression by VNS may therefore depend on the release of NE, a neuromodulator that has anticonvulsant effects. These data suggest that noradrenergic agonists might enhance VNS-induced seizure suppression.     

Quantification of plasma hemoglobin in the presence of bilirubin with bilirubin oxidase

Owing to the overlapping absorption bands of hemoglobin and bilirubin, spectrophotometric determination of plasma free hemoglobin has been plagued by the interference of bilirubin when the latter is present in significant concentrations. To resolve this problem, bilirubin oxidase from Myrothecium verrucaria has been used to remove bilirubin in icteric samples. The enzyme converts bilirubin to biliverdin and eliminates its absorption in the 400 nm region. The absorbance of hemoglobin at 415 nm after Allen baseline correction is used to quantify its concentration. Intra-assay precision of this method on a sample containing 200 mg/L of free hemoglobin and 200 mg/L of bilirubin is 3.1 percent (n = 10), while the between-run precision is 3.8 percent (n = 10). Accuracy studies with samples containing 200 mg/L of bilirubin yielded a straight line: y = 0.90x + 2.31, Sy.x = 3.3, r = 0.99. These results demonstrate that this method can be used to determine free hemoglobin in icteric specimens.     

The diagnosis of brain lesions in newborns with an intrauterine herpetic infection

Enzyme immunoassay and DNA hybridization technique were used to diagnose herpetic infection. Blood and liquor antiherpetic antibodies were detected, with anticytomegalovirus antibodies used as reference ones. Intrauterine herpetic infection was detected in 61.5% of 91 risk group newborns. We should like to emphasize that two laboratory tests should be used to diagnose an intrauterine herpetic infection in newborns. Detection of antiviral antibodies in the CSF is a valuable method for the diagnosis and differentiation of brain involvement in intrauterine herpetic infection.     

Ethanol is a potent inhibitor of canine cerebrospinal fluid production: an acute and reversible effect

Poly(ethylene glycol) and dextran aqueous two-phase systemis (ATPS) were developed to facilitate the separation of components of the proteose peptone fraction of bovine milk, which are mostly large casein derived peptides or glycoproteins. These have proved difficult to purify using conventional chromatographic procedures. ATPS exploit differences in hydrophobicity, size and ionic properties of the proteose peptones with a view to developing methods for future large scale preparations of the individual components of this whey protein fraction.     

Control of glial number: purification from mammalian brain extracts of an inhibitor of astrocyte division

Inhibitors of astrocyte cell division, immunologically related to the sugar moiety of epidermal growth factor receptor and to blood group antigens, have been purified from mammalian brain extracts. Mass spectra, high resolution proton magnetic resonance spectra, and chemical and enzymic analysis of the purified fraction indicated that the compounds isolated were glycosphingolipids, although signals compatible with the presence of aminoacid residues were also observed. Lectin binding indicated the presence of NAc-Neuraminic acid, NAc-glucosamine, fucose, galactose, and glucose. The inhibitor was cytostatic for astrocytes, C6 glioma cells, and endothelial cells, with approximate ID50 of 250 nM. Primary cultures of fibroblasts or 3T3 cells were not affected up to concentrations of 800 nM. Concentrations of the inhibitor of 800 nM or higher, caused non-specific cytotoxicity. The biological and immunological properties of this brain inhibitor were identical to those of the EGF receptor-related inhibitor previously described with the acronym ERI. Because of its source and cytostatic action, the glial inhibitor has been renamed neurostatin. Rabbit antibodies to neurostatin immunostained astrocytes and neurons, both in culture and in tissue sections.     

Fermentation studies of rustmicin production by a Micromonospora sp

The antifungal antibiotic rustmicin was detected in the fermentation broth of the actinomycete MA 7094 as a specific inhibitor of sphingolipid biosynthesis in Candida albicans and as a potent fungicidal agent against Cryptococcus neoformans. Taxonomic characterization by both classical means and PCR fingerprinting supported the assignment of the producing culture to the genus Micromonospora. Fermentation medium optimization studies showed that the concentration of tomato paste in the medium was critical to increased production of rustmicin by MA 7094. The stimulatory effect of tomato paste in the medium on rustmicin production appeared to be related to the maintenance of pH at or below a value of 6.0. Addition of the antifoam agent P-2000 to the fermentation was found to dramatically reduce the rustmicin titer, while substitution of another antifoam agent, UCON-LB625, resulted in a 100% increase in the amount of rustmicin detected. After fermentation optimization studies and the generation of a non-sporulating mutant of MA 7094, the rustmicin titer was increased from an initial titer of 10mg/liter to 145 mg/liter.     

Expression of human cathepsin K in Pichia pastoris and preliminary crystallographic studies of an inhibitor complex

Cathepsin K is a cysteine protease of the papain family, which is predominantly expressed in osteoclasts, and is regarded as a key protease in bone remodeling. To facilitate structural studies of the protein, the wild-type sequence of the protease has been mutated so as to replace a potential N-glycosylation site. We have expressed the mutant human cathepsin K to 190 mg/5 L using the Pichia pastoris expression system. Cathepsin K was inactivated with the mechanism-based inhibitor, APC3328, and crystallized from magnesium formate. A 2.2 A X-ray data set has been collected on crystals belonging to space group P2(1)2(1)2(1), with a = 41.66 A, b = 51.41 A, and c = 107.72 A. There is most likely one molecule per asymmetric unit.     

Possible involvement of phosphatidylinositol 3-kinase in regulated exocytosis: studies in chromaffin cells with inhibitor LY294002

Several lines of evidence suggest that phosphorylated products of phosphatidylinositol play critical functions in the regulation of membrane trafficking along the secretory pathway. To probe the possible involvement of phosphatidylinositol 3-kinase (PI 3-kinase) in regulated exocytosis, we have examined its subcellular distribution in cultured chromaffin cells by immunoreplica analysis and confocal immunofluorescence. We found that the PI 3-kinase heterodimer consisting of the regulatory and catalytic subunits was associated essentially with the subplasmalemmal cytoskeleton in both resting and nicotine-stimulated chromaffin cells. Attempts to immunoprecipitate PI 3-kinase with anti-phosphotyrosine antibodies failed, suggesting that the activity of PI 3-kinase was not modulated by tyrosine phosphorylation and/or physical interaction with SH2-containing proteins in stimulated chromaffin cells. LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a potent inhibitor of PI 3-kinase, produced a dose-dependent inhibition of catecholamine secretion evoked by various secretagogues. Furthermore, cytochemical experiments with rhodamine-labeled phalloidin revealed that LY294002 blocked the disassembly of cortical actin in chromaffin cells stimulated by a depolarizing concentration of potassium. Our results suggest that PI 3-kinase may be one of the important regulatory exocytotic components involved in the signaling cascade controlling actin rearrangements required for catecholamine secretion.     

Potent and selective aromatase inhibitor: in vitro and in vivo studies with s-triazine derivative SEF19

We found a potent aromatase inhibitor through the screening of agents for estrogen-dependent breast cancer. SEF19 (2-(imidazol-1-yl)-4,6-dimorphorino-1,3,5-triazine) decreased 50% of human placental aromatase activity in vitro at the concentration of 5.3 nM. In order to clarify the selectivity of SEF19 for enzyme inhibition, we determined the effect of SEF19 on the activities of four steroidogenic cytochrome P450 enzymes in porcine adrenal gland, P450SCC(side-chain cleavage of cholesterol), P450(11 beta) (11 beta-hydroxylase), P450(17 alpha)(17 alpha-hydroxylase/C17,20 lyase) and P450C21 (21-hydroxylase). SEF19 failed to inhibit the activities of porcine adrenal P450SCC, P450(17 alpha) and P450C21 up to the concentration of 100 microM and showed some inhibition on P450(11 beta) activity at 100 microM, while SEF19 completely nullified the aromatase activity at 1 microM. We also determined the potency of SEF19 for the suppression of aromatase activity in vivo. SEF19 suppressed dose-dependently the uterine hypertrophy of immature rats caused by administration of androstenedione (30 mg/kg, s.c.). The ED50 of SEF19 for the suppression of uterine hypertrophy was 0.8 mumol/kg. These results suggest that SEF19 may serve as a potent and selective agent for the treatment of estrogen-dependent breast cancer.     

Identification of an inhibitor targeting macrophage production of monocyte chemoattractant protein-1 as TGF-beta 1

Monocyte chemoattractant protein-1 (MCP-1) is expressed in a diverse range of cells in response to various pathologic stimuli, whereas little is known about endogenous inhibitors of MCP-1 expression. I sought negative regulators of MCP-1 in culture medium conditioned by several cell lines and found that glomerular mesangial cells exclusively secrete a factor that inhibits expression of MCP-1 by activated macrophages. Treatment of J774.2 macrophages with conditioned medium from mesangial cells blunted the induction of MCP-1 by LPS. Even after the induction, subsequent treatment of macrophages with the conditioned medium down-regulated the MCP-1 expression. Medium conditioned by normal rat glomeruli contained a similar inhibitory activity that was enhanced in regenerating glomeruli, where mesangial cells are activated. The activity of the conditioned medium was not diminished, but enhanced by heat treatment, which was consistent with the unique property of TGF-beta family of molecules. Indeed, the mesangial cell-derived medium contained active TGF-beta 1. An anti-TGF-beta 1 neutralizing Ab abolished the inhibitory effect exerted by the mesangial cell medium, and externally added TGF-beta 1 suppressed the MCP-1 expression by macrophages at both mRNA and protein levels. The inhibitory effect of TGF-beta 1 on MCP-1 was observed in other macrophage cell lines, RAW264.7 and NR8383, and peritoneal macrophages, but not in fibroblastic cell line NRK49F. Treatment of J774.2 macrophages with TGF-beta 1 inhibited LPS induction of c-jun that was found to be crucial for the MCP-1 expression. These data demonstrate that TGF-beta 1 functions as an inhibitor of MCP-1 expression in macrophages possibly via down-regulation of c-Jun/activator protein-1.