Electric and magnetic fields of the brain accompanying internal simulation of movement

We show that the antibacterial agent, (6S)-6-fluoroshikimate, is a substrate for the shikimate transport system of Escherichia coli because in exchange-diffusion experiments it displaced intracellular [14C]shikimate with the same kinetics as did unlabelled shikimate. Other shikimate analogues were also substrates: as judged by similar experiments or, in the case of (6R)-6-fluoroshikimate, by inference.     

Retrobiosynthetic NMR studies with 13C-labeled glucose. Formation of gallic acid in plants and fungi

The biosynthesis of gallic acid was studied in cultures of the fungus Phycomyces blakesleeanus and in leaves of the tree Rhus typhina. Fungal cultures were grown with [1-13C]glucose or with a mixture of unlabeled glucose and [U-13C6]glucose. Young leaves of R. typhina were kept in an incubation chamber and were supplied with a solution containing a mixture of unlabeled glucose and [U-13C6]glucose via the leaf stem. Isotope distributions in isolated gallic acid and aromatic amino acids were analyzed by one-dimensional 1H and 13C NMR spectroscopy. A quantitative analysis of the complex isotopomer composition of metabolites was obtained by deconvolution of the 13C13C coupling multiplets using numerical simulation methods. This approach required the accurate analysis of heavy isotope chemical shift effects in a variety of different isotopomers and the analysis of long range 13C13C coupling constants. The resulting isotopomer patterns were interpreted using a retrobiosynthetic approach based on a comparison between the isotopomer patterns of gallic acid and tyrosine. The data show that both in the fungus and in the plant all carbon atoms of gallic acid are biosynthetically equivalent to carbon atoms of shikimate. Notably, the carboxylic group of gallic acid is derived from the carboxylic group of an early intermediate of the shikimate pathway and not from the side chain of phenylalanine or tyrosine. It follows that the committed precursor of gallic acid is an intermediate of the shikimate pathway prior to prephenate or arogenate, most probably 5-dehydroshikimate. A formation of gallic acid via phenylalanine, the lignin precursor, caffeic acid, or 3,4, 5-trihydroxycinnamic acid can be ruled out as major pathways in the fungus and in young leaves of R. typhina. The incorporation of uniformly 13C-labeled glucose followed by quantitative NMR analysis of isotopomer patterns is suggested as a general method for biosynthetic studies. As shown by the plant experiment, this approach is also applicable to systems with low incorporation rates.     

Effect of auxinhormone lanthanide complexes on the growth of wheat coleoptile

Naphthalene-1-acetic acid (HNAA), dichlorophenoxy acetic acid (HDAA), and indole-3-acetic acid (HIAA) are auxinhormones that can affect the growth of plants. The lanthanide complexes of the above auxinhormone LnA3 x 3H2O (Ln = La3+, Ce3+, Sm3+, Er3+, Yb3+; HA = HNAA, HDAA, HIAA; A = NAA-, DAA-, IAA-) were synthesized and are characterized in this paper. The solubility and IR spectra of these complexes were also studied. Experiments of the effects of LnCl3 x nH2O, HA, and LnA3 x 3H2O on the growth rate of wheat coleoptile sections show, that LnCl3 x nH2O promotes the growth of wheat coleoptile when this compounds concentration is lower than 2 x 10(-5) M and the promotion is very significant when the concentration of Ln3+ is lower than 8 x 10(-6) M. It was also found that the effect of LnA3 x 3H2O on the growth of wheat coleoptile is stronger than that of LnCl3 x nH2O and HA, which indicates that the combination of Ln3+ with HA act synergistically.     

Effect of 6-aminonicotinamide on the pentose phosphate pathway: 31P NMR and tumor growth delay studies

6-aminonicotinamide (6AN) has been shown to enhance radiosensitivity in vitro, although previous in vivo studies failed to show an effect. 31P NMR spectra were obtained by using a one-dimensional chemical shift imaging technique on a first generation transplant of the CD8FI spontaneous mammary carcinoma tumor model. Spectra were obtained both before and 10 h after treatment with 6AN (20 mg/kg). Changes in pH, nucleoside triphosphate/inorganic phosphate, and phosphocreatine/ inorganic phosphate measured at 10 h post-6AN were not significant. A new peak was detected 10 h post-6AN, which was assigned to 6-phosphogluconate (6PG), indicating inhibition of the pentose phosphate pathway (PPP). Based on the spectral data demonstrating inhibition of the PPP at 10 h post-6AN, tumor-bearing mice were irradiated (15 Gy x 3 fractions) on Days 1, 10 or 11, and 21 10 h after administration of 6-aminonicotinamide (20 mg/kg). Tumor-bearing mice receiving 6AN alone (20 mg/kg x 3), radiation alone (15 Gy x 3), or saline were also studied. Tumor growth delay studies indicated that 6AN alone induced a small but significant tumor growth delay (4.3 +/- 0.8 days). Radiation alone induced a tumor growth delay of 34.5 +/- 2.7 days. Treatment with 6AN followed by radiation induced a tumor growth delay of 57.0 +/- 3.8 days. This was significantly greater than the TGD values for treatment with 6AN alone or radiation (P < 0.01). No complete regressions were noted after treatment with 6AN or radiation alone. Concomitant therapy with 6AN plus radiation yielded 6/28 complete regressions (21%), which was significantly greater than radiation (P < 0.05) or 6AN alone (P < 0.01) on this mammary carcinoma.     

Chronic oxytocin pretreatment inhibits adenylyl cyclase activity in cultured rat myometrial cells

To determine whether chronic oxytocin pretreatment inhibits adenylyl cyclase, we compared adenylyl cyclase activity in membranes prepared from cultured, immortalized rat myometrial cells that were untreated or pretreated for 24 h with oxytocin. Chronic oxytocin pretreatment (1 x 10(-5) M for 24 h) attenuated basal, guanosine triphosphate (1 x 10(-5) M)-, isoproterenol (1 x 10(-4) M)-, forskolin (1 x 10(-5) M)-, MnCl2 (20 mM)- or NaF (1 x 10(-2) M)-stimulated adenylyl cyclase activity by 27 +/- 5% to 39 +/- 11% (n = 6, p < 0.05). Oxytocin pretreatment for 2 h (n = 5) did not produce a significant effect. To understand the mechanism by which oxytocin pretreatment decreased activity of the adenylyl cyclase pathway, we compared effects of pretreatment with either oxytocin or phenylephrine on adenylyl cyclase activity and determined the effects of Gi inhibition and protein kinase C (PKC) depletion. Chronic (24 h) phenylephrine pretreatment (1 x 10(-4) M) had effects similar to those of oxytocin pretreatment (1 x 10(-5) M). PKC depletion with phorbol 12-myristate 13-acetate (1 x 10(-6) M, 41 h) prevented attenuation of adenylyl cyclase activity by oxytocin pretreatment (1 x 10(-5) M for 24 h). Inhibition of Gi by pertussis toxin pretreatment (1.25 microg/ml, 41 h) had no significant effect. These findings suggest that chronic oxytocin pretreatment desensitizes the adenylyl cyclase pathway by a cross-regulatory mechanism that involves activation of Gq and PKC.     

Stimulation of tetrapyrrole synthesis in mammalian epithelial cells in culture by exposure to aminolaevulinic acid

Tetrapyrrole synthesis in CNCM-1221 cells exposed to 0.6 mM aminolaevulinic acid (ALA) was found to be approximately linear over a 6-h period of incubation. The rate was not significantly affected by cell density over a range of 0.015 to 0.15 x 10(6) cells cm(-2) (final cell density). Tetrapyrrole synthesis was not affected by GABA or glutamic acid in concentrations up to 6 mM and 2.72 mM respectively, suggesting that these amino acids, which are similar in structure to ALA, do not competitively inhibit the ALA uptake pathway in these cells. Pre-exposure to haem arginate (up to 100 microM) was inhibitory, presumably by suppression (through the inhibition of ALA synthase) of an endogenous component of the response. The ALA-stimulated response was not modified by co-exposure to AIA (up to 100 mg ml(-1)). Despite significant reduction of protein synthesis, the porphyrinogenic response of cells exposed to ALA was unaffected by cycloheximide (10 microg ml(-1)) or actinomycin D (10 microg ml(-1)) even when cells were preincubated with these agents for 3 h before ALA exposure. Fetal bovine serum (10%) inhibited tetrapyrrole synthesis by 30% but increased the rate of porphyrin export by cells by a factor of 1.5. The uptake of [14C]ALA was shown to be strongly influenced by the density of the cultures. In dense cultures (final cell density of approximately 0.15 x 10(6) cells cm(-2)), the ALA uptake rate was less than 0.8 compared with a maximum rate of 4.2 fmol per cell h(-1) at a cell density of 0.02 x 10(6) cells cm(-2). Since tetrapyrrole synthesis is less affected than ALA uptake by cell density, the resultant discrepancy in ALA incorporation occurring in dense cultures implies that endogenous ALA synthesis is induced in these cells. ALA uptake was not affected by cycloheximide or actinomycin D in serum-free conditions. However, fetal bovine serum decreased external ALA uptake by about 50%. This effect was abrogated by preincubation with cycloheximide.     

Prospective evaluation of external ocular microbial growth and aqueous humor contamination during cataract surgery

Primary cultures of porcine brain capillary endothelial cells grown on collagen coated polycarbonate membranes were used to build up an in vitro-model for the blood-brain barrier. Improved cultivation techniques allowed cell-storage and experiments under serum-free conditions. We employed this model to perform permeability studies in vitro with the radioactively labelled marker substances sucrose, retinoic acid, retinol, haloperidol, caffeine, and mannitol. Permeability values obtained with this blood-brain barrier model (1. 0x10-6 cm/s for sucrose, 6.2x10-6 cm/s for retinoic acid, 4.8x10-6 cm/s for retinol, 49.5x10-6 cm/s for haloperidol, 62.4x10-6 cm/s for caffeine, and 1.8x10-6 cm/s for mannitol) show a good correlation to data which are already known from in vivo-experiments. As judged by the sucrose permeability our blood-brain barrier model is less permeable than numerous other models published so far. Therefore it represents a powerful tool for in vitro-prediction of blood-brain barrier permeability of drugs and offers the possibility to scan a large quantity of drugs for their potential to enter the brain.     

Protective effect of the insulin-like growth factor I receptor on apoptosis induced by okadaic acid

Okadaic acid (OKA), a potent inhibitor of serine phosphatases at concentrations as low as 20-25 nM, induces apoptosis of R- mouse embryo fibroblasts, which are 3T3-like cells devoid of type 1 insulin-like growth factor receptors (IGF-IRs). From R- cells, we have generated (by stable transfection) cell lines with IGF-IR numbers ranging from 0 (R- cells) to >10(6) receptors per cell. The wild-type IGF-IR protects R- cells from OKA-induced apoptosis, its protective effect being exquisitely dependent on the number of receptors. A small increment in wild-type receptor number (from 15 x 10(3) to 22 x 10(3) receptors/cell) is sufficient to change R(-)-derived cells from sensitive to resistant to apoptosis. We have also studied the effect of various mutations of the IGF-IR on its ability to protect R(-)-derived cells from OKA-induced apoptosis. Our data indicate a correlation between protection from apoptosis and the ability of the receptor to respond to insulin-like growth factor I with mitogenesis.     

Alcohol inhalation in asthma therapy

As part of the search for anticomplementary active components from natural products, the anticomplementary properties of methanolic extracts from the flower buds of Magnoliafargesii have been investigated. Bioassay-guided chromatographic separation of the active constituents led to the isolation of compound 1, whose structure was identified by spectroscopic methods to be kaempferol 3-O-beta-D-(6"-O-coumaroyl)glucopyranoside (tiliroside). Tiliroside showed very potent anti-complement activity (IC50=5.4 x 10(-5) M) on the classical pathway of the complement system, even higher than rosmarinic acid, which is a well-known inhibitor against the complement system. On the other hand, the hydrolysates of tiliroside, kaempferol, astragalin and p-coumaric acid showed very weak activity on this system.     

Endothelin-1 inhibits L-type Ca2+ current enhanced by isoprenaline in rat atrial myocytes

Endothelin-1 (ET-1) was shown to exert direct cardiac effects by complex signaling pathways and to interact with neurotransmitter regulation of cardiac activity. The effect of ET-1 was investigated on the beta-adrenergic stimulation of cardiac L-type Ca2+ current (ICaL) on isolated rat atrial myocytes by using the patch-clamp technique. ET-1 (5 x 10(-8) M) reversed the increase in ICaL induced by isoprenaline (10(-6) M) but had no effect on basal ICaL and on (-) Bay K 8644-increased ICaL (10(-6) M); so ET-1 might exert an effect only when the Ca2+ channels are phosphorylated. The antiadrenergic action of ET-1, blocked by BQ-123 (10(-6) M) and unaffected by IRL 1038 (3.5 x 10(-8) M) should be mediated by ET-A receptors. The inhibitory action of ET-1 was still observed when ICaL was previously increased by forskolin (3 x 10(-6) M), 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP; 200 microM), or cAMP (100 microM) in presence of isobutyl methyl xanthine (IBMX; 10(-6) M), suggesting that the antiadrenergic action of ET-1 on ICaL was exerted independent of the cAMP-dependent phosphorylation pathway. ET-1 is known to be an activator of phosphoinositide hydrolysis, resulting in an increased production of IP3 and diacylglycerol (DAG). A Ca(2+)-dependent inhibition of ICaL consequently to an elevation of the intracellular Ca2+ pool via IP3 might be excluded in the action of ET-1, because of the presence of EGTA in the intrapipette medium. ET-1 reversed the isoprenaline-induced increase in ICaL in the presence of protein kinase C inhibitor [PKC(19-31); 100 microM), making unlikely the involvement of a DAG-dependent activation of PKC. Therefore the antiadrenergic action of ET-1 might also be independent on the phosphoinositide pathway.     

Growth of single HL60 cells in liquid culture: analysis of the influences of differentiative agents

Treatment of serum-free grown HL60 cells with certain combined amounts of retinoic acid (9-cis or all-trans RA) and 1 alpha 25 dihydroxyvitamin D3 (D3) results in differentiation of 71-77% of cells towards either neutrophils or monocytes. Studies of the differentiation of HL60 cells in flask cultures does not reveal: (i) the extent to which selective growth of cells might have occurred; and (ii) the overall level of cell survival. This information can be obtained by monitoring the effects of differentiative agents on individual cells. Serum-free grown HL60 cells were cultured as single cells in microtitre wells in conditioned medium obtained from exponentially growing and serum-free cultures of HL60. This resulted in a cloning efficiency of 85% and HL60 cells doubled every 24 h. During a period of exponential growth < 0.5 to 2% of the cells generated died. Single HL60 cells were treated with 9-cis and all-trans RA (5 x 10(-7) M) together with a small amount of D3 (3.9 x 10(-14) M) to promote neutrophil differentiation. D3 alone (10(-7) M) and D3 (5 x 10(-9) M) in combination with 9-cis RA (10(-8) M) were used to promote monocyte differentiation. The growth kinetics of HL60 cell cultures that were differentiating to neutrophils and to monocytes were similar. Single-cell experiments have revealed that: (i) differentiating HL60 cells undergo a variable number of divisions (two to five) prior to arresting their growth; and (ii) up to 33% of the cells that are generated (by day 5) die. Seventy to eighty per cent of the cells in each of the wells had matured. These findings have important implications in regard to whether retinoids and D3 provide signals that determine the choice of maturation pathway or that merely facilitate selective survival and/or expansion of cells that have independently determined their differentiation fates.     

Purification and preliminary characterization of a serine hydrolase involved in the microbial degradation of polychlorinated biphenyls

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA) hydrolase (BphD), an enzyme of the biphenyl biodegradation pathway encoded by the bphD gene of Burkholderia cepacia LB400, was hyperexpressed and purified to apparent homogeneity. SDS-polyacrylamide gel electrophoresis confirmed that BphD has a subunit molecular mass of 32 kDa, while gel filtration demonstrated that it is a homotetramer of molecular weight 122,000. The enzyme hydrolyzed 6-phenyl-HODA with a kcat of 5.0 (+/- 0.07) s-1 and a kcat/Km of 2.0 (+/- 0.08) x 10(7) M-1 s-1 (100 mM phosphate, pH 7.5, 25 degreesC). The specificity of BphD for other 2-hydroxy-6-oxohexa-2,4-dienoates (HODAs) decreased markedly with the size of the C6 substituent; 6-methyl-HODA, the meta cleavage product of 3-methylcatechol, was hydrolyzed approximately 2300 times less specifically than 6-phenyl-HODA. By comparison, the homologous hydrolase from the toluene degradation pathway, TodF, showed highest specificity for 6-methyl- and 6-ethyl-HODA (kcat/Km of 2.0 (+/- 0.05) x 10(6) M-1 s-1 and 9.0 (+/- 0.5) x 10(6) M-1 s-1, respectively). TodF showed no detectable activity toward 6-phenyl-HODA and 6-tert-butyl-HODA. Neither BphD nor TodF hydrolyzed 5-methyl-HODA efficiently. The kcat of BphD determined by monitoring product formation was about half that determined by monitoring substrate disappearance, suggesting that some uncoupling of substrate utilization and product formation occurs during the enzyme catalyzed reaction. Crystals of BphD were obtained using ammonium sulfate combined with polyethylene glycol 400 as the precipitant. Diffraction was observed to a resolution of at least 1.9 A, and the evaluation of self-rotation functions confirmed 222 (D2) molecular symmetry.     

Canine jugular vein endothelial cell monolayers in vitro: vasomediator-activated diffusive albumin pathway

Cultured canine jugular vein endothelial cells were seeded on polycarbonate filters to create an in vitro permeability assay. The calculated diffusive permeability coefficient for FITC-BSA across untreated monolayers was 1.1 +/- 0.4 x 10(-6) cm/s. After 15-min incubations with either histamine or bradykinin, the resistance to albumin flux across the monolayers was reduced significantly. Diffusive albumin permeability coefficients were 3.4 +/- 1.8 x 10(-6) and 4.1 +/- 2.0 x 10(-6) cm/s, respectively. Ultrastructural morphometric analyses of the endothelial cell monolayers served to define uniform dimensions of intercellular clefts and similar plasmalemmal vesicle densities in the untreated and the vasomediator-activated monolayers. These results are consistent with the interpretation that the vasomediator-activated pathway across the venous endothelial monolayers is not dependent on sustained intercellular gap formation or sustained expansion of the plasmalemmal vesicle population for the 15-min observation periods. Whether the increased albumin flux is dependent on transient gap formation or on physical changes within the venous endothelial cell glycocalyx remains to be tested.     

Expression of a functional endothelin (ETA) receptor in human meningiomas

Endothelin (ET) receptor subtypes (ETA and ETB) in human meningiomas were characterized using quantitative receptor autoradiography. A single class of high-affinity 125I-ET-1 binding sites was localized in all meningioma tissue studied (dissociation constant: 2.4 +/- 0.3 nM, maximum binding capacity: 319 +/- 66 fmol/mg (mean +/- standard error of the mean for 13 tumors)). Unlabeled ET-1 showed a strong affinity for 125I-ET-1 binding to tissue sections of the tumors with a 50% inhibiting concentration (IC50) of 2.9 +/- 0.7 x 10(-9) M, whereas ET-3 showed a much lower affinity (IC50: 8.4 +/- 2.5 x 10(-6) M). Sarafotoxin S6c, a selective agonist for the ETB receptor, could not compete for 125I-ET-1 binding to meningiomas. Endothelin-1 significantly stimulated deoxyribonucleic acid (DNA) synthesis in a dose-dependent manner in cultured human meningioma cells. In contrast, no significant stimulation of DNA synthesis occurred with an S6c concentration up to 10(-7) M. Pretreatment of the meningioma cells with pertussis toxin, a bacterial toxin that adds adenosine 5'-diphosphate-ribose to the alpha subunit of guanine nucleotide binding (G) proteins such as Gi or G(o), induced a concentration-dependent reduction in ET-stimulated DNA synthesis in meningioma cells, but did not affect the epidermal growth factor-induced DNA synthesis. These observations suggest that the ETA receptor is predominantly expressed in human meningioma tissue and that ET may act as a growth factor on the meningioma cells by interacting with the ETA receptor and by pertussis toxin-sensitive mechanisms.     

Linoleic acid induces relaxation and hyperpolarization of the pig coronary artery

Linoleic acid, a polyunsaturated C18 fatty acid, is one of the major fatty acids in the coronary arterial wall. Although diets rich in linoleic acid reduce blood pressure and prevent coronary artery disease in both humans and animals, very little is known about its mechanism of action. We believed that its beneficial effects might be mediated by changes in vascular tone. We investigated whether linoleic acid induces relaxation of porcine coronary artery rings and the mechanism involved in this process. Linoleic acid and two of its metabolites, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-hydroperoxyoctadecadienoic acid (13-HPODE), induced dose-dependent relaxation of prostaglandin (PG) F2alpha-precontracted rings that was not affected by indomethacin (10[-5] mol/L), a cyclooxygenase inhibitor, or cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC; 10[-5] mol/L), a lipoxygenase inhibitor. Removal of endothelial cells had no effect on vasorelaxation, suggesting a direct effect on the vascular smooth muscle cells (VSMC). When rings were contracted with KCl, linoleic acid failed to induce relaxation. Although tetrabutylammonium (5 x 10[-3] mol/L), a nonselective K+ channel blocker, slightly inhibited the relaxation caused by linoleic acid, glibenclamide (10[-6] mol/L), an ATP-sensitive K+ channel blocker, and charybdotoxin (7.5x10[-8] mol/L) or tetraethylammonium (5x10[-3] mol/L), two different Ca2+-activated K+ channel blockers, had no effect. However, relaxation was completely blocked by ouabain (5x10[-7] mol/L), a Na+/K+-ATPase inhibitor, or by a K+-free solution. In addition, linoleic acid (10[-6] mol/L) caused sustained hyperpolarization of porcine coronary VSMC (from -49.5+/-2.0 to -60.7+/-4.2 mV), which was also abolished by ouabain. We concluded that linoleic acid induces relaxation and hyperpolarization of porcine coronary VSMC via a mechanism that involves activation of the Na+/K+-ATPase pump.     

In vitro evolution of horse heart myoglobin to increase peroxidase activity

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with k1 (for H2O2 oxidation of metmyoglobin) of 1. 34 x 10(4) M-1 s-1 ( approximately 25-fold that of wild-type myoglobin) and k3 [for reducing the substrate (2, 2'-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 x 10(6) M-1 s-1 (1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the Tm of the quadruple variant is decreased only slightly compared with wild-type (74.1 degreesC vs. 76.5 degreesC). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (kon, 22.2 x 10(-6) M-1 s-1 vs. 22.3 x 10(-6) M-1 s-1; koff, 24.3 s-1 vs. 24.2 s-1; KO2, 0.91 x 10(-6) M-1 vs. 0.92 x 10(-6) M-1). The affinity of the quadruple variant for CO is increased slightly (kon, 0.90 x 10(-6) M-1s-1 vs. 0.51 x 10(-6) M-1s-1; koff, 5.08 s-1 vs. 3.51 s-1; KCO, 1.77 x 10(-7) M-1 vs. 1.45 x 10(-7) M-1). All four substitutions are in the heme pocket and within 5 A of the heme group.     

Lifetime comorbidity, lifetime history of psychosis and suicide attempts, and current symptoms of patients with deteriorated affective disorder

Expression of insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose-6-phosphate (IGF-II/Man6P) receptors in cultured bovine alveolar macrophages (BAM) was demonstrated by competitive binding studies and crosslinking experiments. Western blotting of protein extracts from cultured BAM using an anti bovine IGF-II/Man6P receptor antiserum (#66416) confirmed the presence of IGF-II/Man6P receptors on BAM. The effects of IGFs and Man6P on generation of inositol phosphates was measured by HPLC analysis of perchloric acid extracts from myo-[3H]inositol-labelled cultured BAM. IGF-I at nanomolar concentrations and Man6P (10[-8]-10[-3] M) stimulated the accumulation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 after 30 sec. IGF-II (up to 2.3 x 10[-8] M) had no significant effect on inositol phosphate accumulation under the same conditions. Both IGFs and Man6P induced a rise in [Ca2+]i in cultured BAM. In addition, using the fluorescent dye SNARF-1/AM we could demonstrate rapid but small IGF-II (10[-9] M) triggered acidification (0.07 pH units) of cultured BAM. Taken together, our results indicate not only the presence of both IGF-I and IGF-II/Man6P receptors on BAM, but also provide evidence of the linkage of the IGF-I receptor to the inositol phosphate system.     

Involvement of cerulospinal glutamatergic neurotransmission in fentanyl-induced muscular rigidity in the rat

BACKGROUND: Investigators in the authors' laboratory previously established the critical participation of the cerulospinal noradrenergic pathway in muscular rigidity elicited by fentanyl. The identification of colocalization of glutamate with tyrosine hydroxylase in most locus ceruleus neurons suggests a role for cerulospinal glutamatergic neurotransmission in fentanyl-induced muscular rigidity. This suggestion and the subtype(s) of glutamate receptors involved were investigated here. METHODS: Electromyographic signals activated by bilateral microinjection of 2.5 microg fentanyl into the locus ceruleus were recorded differentially from the left sacrococcygeus dorsi lateralis muscle of adult male Sprague-Dawley rats. The effect of intrathecal administration at the lower lumbar spinal cord of various N-methyl-D-aspartate (NMDA) and non-NMDA receptor antagonists or agonists on this index of muscular rigidity was studied. Rats were under mechanical ventilation, and intravenous infusion of ketamine (30 mg x kg(-1) x h(-1)) was maintained until 10 min before fentanyl was administered. RESULTS: Microinjection of fentanyl bilaterally into the locus ceruleus increased the root mean square and decreased the mean power frequency values of electromyographic signals. The efficacy of fentanyl to elicit muscular rigidity in this manner was significantly reduced by previous intrathecal administration of either 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), D-(-)-2-amino-5-phosphonovaleric acid (AP5), or (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). Intrathecal administration of kainic acid or NMDA also resulted in significant electromyographic activation. CONCLUSIONS: In addition to the cerulospinal noradrenergic mechanism, the cerulospinal glutamatergic pathway and both NMDA and non-NMDA receptors in the spinal cord may mediate fentanyl-induced muscular rigidity in the rat.     

Molecular mode of action of the antifungal beta-amino acid BAY 10-8888

BAY 10-8888 is a cyclic beta-amino acid that is related to cispentacin and that has antifungal activity. Candida albicans cells accumulated BAY 10-8888 intracellularly to a concentration about 200 that in the medium when grown in media with a variety of nitrogen sources. In complex growth medium, BAY 10-8888 transport activity was markedly reduced and was paralleled by a decrease in its antifungal activity. Uptake of BAY 10-8888 was mediated by an H+-coupled amino acid transporter with specificity for branched-chain amino acids (isoleucine, leucine, and valine) and showed a KT (Michaelis constant of the transport reaction) of 0.95 mM and a Vmax of 18.9 nmol x min-1 x 10(7) cells-1. Similar to the transport of natural amino acids in Saccharomyces cerevisiae, the transport of BAY 10-8888 into the cell was unidirectional. Efflux occurred by diffusion and was not carrier mediated. Inside the cell BAY 10-8888 inhibited specifically isoleucyl-tRNA synthetase, resulting in inhibition of protein synthesis and cell growth. Intracellular isoleucine reversed BAY 10-8888-induced growth inhibition. BAY 10-8888 was not incorporated into proteins. BAY 10-8888 inhibited isoleucyl-tRNA synthetase with the same concentration dependency as protein biosynthesis in intact cells assuming 200-fold accumulation.     

The effects of reverse micelles on the reaction mechanism of alpha-chymotrypsin

Reverse micelles were employed to test the accuracy of the widely accepted mechanism for alpha-chymotrypsin in a highly structured aqueous system similar to intracellular conditions. Results yielded from spectrophotometrical assays of the alpha-chymotrypsin catalyzed hydrolysis of both p-nitrophenyl acetate (p-NPA) and p-nitrophenyl trimethylacetate (p-NPTA) were kinetically analyzed to determine constants typical of the proposed mechanistic model. This was accomplished through the establishment of a control, i.e. the well studied buffer system, for comparison between the reverse micellular environment and a bulk aqueous solution. Control group results yielded kinetic constants in favor of the proposed mechanism (Km = 1.55 x 10(-5) +/- 1.40 x 10(-6) M for p-NPA and a Km = 4.97 x 10(-6) +/- 2.29 x 10(-7) M, Km(app) = 4.92 x 10(-6) +/- 2.33 x 10(-8) M, k2 = 4.34 x 10(-3) +/- 1.31 x 10(-3), k(cat) = 1.96 x 10(-3) +/- 2.47 x 10(-4), and Ks = 1.60 x 10(-5) +/- 4.61 x 10(-6) M for p-NPTA). In contrast, similar reactions of the enzyme in a reverse micellular system produced kinetic constants atypical to that representative of the textbook mechanism. (Km = 1.59 x 10(-4) +/- 2.70 x 10(-5) M, Ks = -8.67 x 10(-5) +/- 4.46 x 10(-5) M and Km(app) = -4.80 x 10(-5) +/- 7.05 x 10(-5) M for p-NPA and Km = 1.95 x 10(-4) +/- 9.28 x 10(-5) M, Km(app) = -1.79 x 10(-4) +/- 2.36 x 10(-5) M, and Ks = -3.95 x 10(-4) +/- 1.18 x 10(-4) M for p-NPTA). In addition to negative kinetic constants, alpha-chymotrypsin seemed to display characteristics indicative of super-activity and a hysteretic response. Overall, the widely accepted mechanism for alpha-chymotrypsin appeared to fail within the confines of reverse micelles, due to the direct influence of the system's highly structured form.