The pyridoindole antioxidant stobadine inhibited glycation-induced absorbance and fluorescence changes in albumin

We studied the effect of the pyridoindole antioxidant stobadine on glycation-induced absorbance and fluorescence changes in bovine serum albumin (BSA), used as a model protein. Incubation of BSA (4 mg/ml) with glucose (100–400 mM) in 0.12 M phosphate buffer, pH 7.4, in the presence of 100 M Cu²⁺ at 37°C resulted in a time-dependent increase of absorbance (320 nm) and fluorescence (excitation 350 nm, emission 415 nm). The process was found to be dependent on the presence of oxygen and transition metal ions, but equimolar iron could not fully substitute for the activity of copper. The glucose-induced chromo- and fluorophore formation was reduced significantly by stobadine. For 200 mM glucose, in 7- and 14-day incubations, 51%–60% inhibition was obtained at a stobadine concentration of 0.1 mM, and the effect leveled off at higher concentrations of the drug. No inhibition was observed withN-acetyl stobadine, a derivative with restricted antioxidant activity. Since stobadine did not affect the Amadori product formation determined by the thiobarbituric acid (TBA) method as 5-hydroxymethyl furfural (5-HMF) released in boiling oxalic acid, the inhibitory action of stobadine may be explained by its interference with metal-catalyzed oxidation reactions following after the glycation step. The results obtained suggest that antioxidant therapy could be used to limit the damage from adverse glycation-induced processes in diabetes mellitus.     

内毒素血症狗肝脏蛋白激酶A动力学特点的变化

静脉注射内毒素后4h,狗肝脏Ⅱ型蛋白激酶A活力显著低于对照组,对酶激活物CAMP以及酶反应底物ATP和组蛋白的最大反应速度显著降低,米氏常数无变化;狗肝脏Ⅰ型蛋白激酶A活力、最大反应速度及米氏常数均无变化。结果提示,狗内毒素血症时肝脏Ⅱ型蛋白激酶A活性受抑。     

The early internal vascularization of the rat brain

Summary The internal vascularization of the brain was studied in foetuses of normal and protein-deprived rats from embryonic day (E) 12 to 15. The position of vascular branches showed distinct relations to the various zones of the neuroepithelium. The possibility that various parts of the vascular system may differ in function, maturation, and morphogenetic relations to the neuroepithelium must be considered. The distinct vascular layers were therefore given names relating them to the respective wall zone. The ingrowth of straight stem vessels from the epiparenchymal vascular plexus into the neuroepithelium and the formation of vascular branches close to the ventricular system were referred to as stage I of the internal vascularization. The resulting plexus was called the deep vascular plexus of the ventricular zone. Its formation followed the same temporospatial gradients as the formation of the marginal zone. Following the formation of the intermediate zone, more stem vessels entered the neuroepithelium and a superficial vascular plexus of the ventricular zone was formed (stage II). This plexus was positioned close to the border between the ventricular zone and the intermediate zone. Subsequently, vascular branches also formed plexuses of the intermediate and subventricular zones (stage III). No intraepithelial vessels were seen on E 12. The temporospatial gradients in the telencephalic vesicles were caudal to rostral and lateral to medial, starting in the parts corresponding to the ganglionic eminence in the floor of the lateral ventricle on E 13. Only the dorsomedial angles of the hemispheres showed no vessels on E 15.No obvious differences were seen between the normal and the protein-deprived foetuses regarding the timing and extent of vascularization or the size and appearance of wall zones in the immature central nervous (I-CNS).     

A comparative view of Rickettsia tsutsugamushi and the other groups of rickettsiae

Recent researches on the rickettsial group microorganisms are summarized in their comparative aspects of morphology, cultivation and multiplication, susceptibility to chemotherapeutics, chemical structure of envelopes, nucleic acid, protein constitution, and gene structures. From this overview, Rickettsia tsutsugamushi seems to have different properties from the others and should be reclassified into a new genus, and a new species name as Orientia tsutsugamushi is proposed.Presented at the 4th International Symposium on Rickettsiae and Rickettsial Diseases, Pietany, C.S.F.R., 1–6 October, 1990.     

Inhibition of Ca2+ transport associated with cAMP-dependent protein phosphorylation in rat cardiac sarcoplasmic reticulum by triorganotins

Organotin compounds have been shown to interfere with cardiovascular system. We have studied the in vitro and in vivo effects of tributyltin bromide (TBT), triethyltin bromide (TET) and trimethyltin chloride (TMT) on the cardiac SR Ca²⁺ pump, as well as on protein phosphorylation of SR proteins, in order to understand the relative potency of these tin compounds. All the three tin compounds inhibited cardiac SR⁴⁵Ca uptake and Ca²⁺-ATPase in vitro in a concentration-dependent manner. The order of potency for Ca²⁺-ATPase as determined by IC₅₀, is TBT (2 M) > TET (63 M) > TMT (280 M). For⁴⁵Ca uptake, it followed the same order i.e., TBT (0.35 M) > TET (10 M) > TMT (440 M). In agreement with the in vitro results, both SR Ca²⁺-ATPase and⁴⁵Ca uptake were significantly inhibited in rats treated with these tin compounds, indicating that these tin compounds inhibit cardiac SR Ca²⁺ transport. cAMP significantly elevated (70–80%) the³²P-binding to SR proteins in vitro in the absence of any organotin. In the presence of organotins, cAMP-stimulated³²P-binding to proteins was significantly reduced, but the decrease was concentration dependent only at lower concentrations. The order of potency is TBT > TET > TMT. In agreement with in vitro studies, cAMP-dependent³²P bound to proteins was significantly reduced in rats treated with TBT, TET and TMT. SDS-polyacrylamide gel electrophoresis of the cardiac SR revealed at least 30 Coomassie blue stainable bands ranging from 9 to 120 kDa. Autoradiographs from samples incubated in the presence of cAMP indicated³²P incorporation in seven bands. Of these, the band corresponding to about 24 kDa molecular weight protein decreased in its intensity with the treatment of organotins. These results suggest that triorganotins may be affecting Ca²⁺ pumping mechanisms through the alteration of phosphorylation of specific proteins in rat cardiac SR.This work has been presented in part at the Annual meeting of Society of Toxicology, 1990 at Miami Beach, FL. The Toxicologist 10: 35 & 108 (1990).     

Phospholipase D in heart: basal activity and stimulation by phorbol esters and aluminum fluoride

Summary Evidence for a general role of phospholipase D in signal transduction is accumulating. In the present study, the activity of the enzyme was investigated in heart tissue under basal conditions and after addition of phorbol esters or aluminum fluoride (AlF _inf4 ^sup– ; 10 mM NaF plus 10 M AlCl₃). Atria of rats and chickens were incubated with [³H]-myristic acid in order to label preferentially phosphatidylcholine. Under basal conditions, the tissues generated choline and phosphatidic acid (PtdOH), the primary catalytic products of phospholipase D. When 0.5 or 2.0% ethanol was present, [³H]-phosphatidyl-ethanol (PETH) was rapidly formed at the expense of [³H]-PtdOH. This transphosphatidylation reaction is specific for phospholipase D activity. The basal formation of PETH was not inhibited by a Ca²⁺-free, EGTA-containing medium. - The phorbol ester 4-phorbol-12,13-dibutyrate (PDB), which is known to activate protein kinase C, enhanced the net formation of choline, whereas the inactive 4-phorbol-13-acetate (PAc) was ineffective. PDB (0.2 M), in contrast to PAc, also increased the formation of [³H]-PtdOH and, in the presence of ethanol, of [³H]-PETH. The PDB-evoked formation of PETH occurred again at the expense of PtdOH. Treshold and maximum effective concentrations of PDB were 10 nM and 0.2–0.6 M, respectively. The effects of PDB on either choline efflux and generation of PETH showed the same Ca^t+-dependency, i.e., both effects were blocked by a Ca²⁺-free, EGTA-containing medium, but not by a Ca²⁺-free medium without EGTA. In protein kinase C-deficient tissue which was prepared by pretreatment with 0.61 M PDB for 27 h, PDB failed to enhance the formation of PtdOH and PETH. - A1F4–, a known activator of G-proteins, increased not only the tissue content of inositol phosphates, but also markedly enhanced choline efflux and formation of [³H]-PtdOH and PETH. In conclusion, in mammalian and avian atria a high phospholipase D activity was found even under basal conditions. The enzyme was stimulated by protein kinase C and presumably by a G protein.Abbreviations IP inositol phosphate - DAG diacylglycerol - PL phospholipase - PtdOH phosphatidic acid - PETH phosphatidylethanol - PDB 4-phorbol-12,13-dibutyrate - PAc 4-phorbol-13-acetate - AlF _inf4 ^sup– aluminum fluoride - DMSO dimethylsulfoxide Correspondence to K. Löffelholz at the above address     

KT/V and protein catabolic rate determination from serial urea measurement in the dialysate effluent stream

A bloodless technique of evaluating protein catabolic rate (PCR) and KT/V (K, clearance; T, dialysis time; V, urea distribution volume) in hemodialysis patients is presented based on serial measurement of urea in the dialysate effluent stream. PCR follows from equating urea generation and urea removal over a 7 day cycle, changes in body stores being comparatively negligible: PCR = 0.026 [U1 + U2 + U3]/BWdry + 0.17, where U1 is the amount of urea in mmol appearing in the dialysate for each session in the 7 day period. KT/V is obtained from the slope of the natural logarithm of spent dialysate urea concentration-time plot: KT/V = [- slope.T + 3.delta BW/BWdry]/[1 - 0.01786.T(hr], where delta BW = amount ultrafiltered in liters. The dialysate-based approach was validated and compared with conventional urea kinetic modeling (UKM) for 17 patients studied for three consecutive dialyses. The dialysate-based and UKM values of PCR agreed well when in vivo clearance values based on total dialysate collection were used for UKM. KT/V values agreed poorly on a session-by-session basis but were nearly equivalent when averaged for the three dialyses of the week. These findings lay the foundation for UKM automation with a urea sensor in the effluent dialysate stream.     

Human acoustic neurinomas: Nervous system specific biochemical parameters

Summary A series of 24 human acoustic neurinomas from 24 patients has been assayed for several biochemical parameters characteristic of the nervous system. S 100 protein, 2, 3-cyclic nucleotide 3-phosphohydrolase activity, and the myelin lipids galactosylceramide and sulfogalactosylceramide (sulfatide). Myelin basic protein was not detected. These findings further support the neuroectodermal origin of the human acoustic neurinoma, and provide additional biochemical markers for further study.     

Lipid and protein histochemistry of enamel — Effects of fluoride

Summary Staining reactions for a number of histochemical procedures for lipophilic staining and protein were studied in the enamel matrix along the length of rat incisors. Sudan Black gave a positive stain across the whole thickness of very early enamel (up to 30 m) but this staining only continued as a narrow band close to the ameloblasts as the enamel matured. A variety of tests for protein produced almost identical staining patterns in enamel matrix up to 100 m thick. Since the pattern of lipid staining persisted, after using a number of procedures which could normally be expected to remove lipid, it is suggested that Sudan Black positive staining may be due to lipophilic protein rather than lipid itself. Fluoride did not significantly alter the staining reactions for lipid and protein but did proceduce matrix which was much more effectively stained by cross-linking agents FFDNB and FF sulphene.     

Steady-state kinetics of imipramine in patients

Steady-state plasma level kinetics were studied in 76 patients given imipramine (IP) 150 to 225 mg/day for 2–5 weeks. IP was given in three divided doses at 8.00 a.m., 1.00 p.m. and 5.00 p.m. Plasma concentrations of IP and its active metabolite desipramine (DMI) were determined by quantitative in situ thin-layer chromatography. The plasma levels of IP and DMI showed pronounced flucutations throughout the day with a ratio of about 2 between highest and lowest level. Patients with steady-state levels of IP and/or DMI below 50 g/l reached this within 1 week of treatment. Patients with higher steady-state levels reached steady-state concentrations within 2–3 weeks. There were some intraindividual fluctuations in plasma levels from week to week after steady state had been reached (coefficient of variation: 10–20%). Interindividually, the steady-state levels corrected to a dose of 3.5 mg/kg per day varied considerably: IP: 6–356 g/l, DMI: 24–659 g/l and IP+DMI: 58–809 g/l. The steady-state plasma levels showed a skew distribution that became normal by logarithmic transformation. The IP/DMI ratio ranged from 0.07 to 5.5 with a median value of 0.47. Compared to data from amitriptyline treated patients the IP/DMI ratios had significantly lower median value and larger variation than the corresponding plasma level ratios of amitriptyline/nortriptyline. Several statistically significant differences in steady-state levels between age groups were found. For IP: Women aged 30–39 had lower levels than women aged 20–29, 40–49, and 50–59, and men aged 50–59 and 60–65; men aged 30–39 had lower levels than men aged 60–65. For DMI: Women aged 30–39 had lower levels than women aged 50–59.