An Improved HPLC-Electrochemical Detection Method for Measuring Brain Levels of 5-S-Cysteinyldopamine, 5-S-Cysteinyl-3,4-Dihydroxyphenylalanine, and 5-S-Cysteinyl-3,4-Dihydroxyphenylacetic Acid

Brain levels of the 5-S-cysteinyl adducts of 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), and dopamine were determined in several mammalian species. The low levels of the compounds and the risk of artifacts during sample preparation necessitated rather profound modifications of the assaying method. The refined method has made it possible to present more accurate data than those previously reported from this laboratory. The occurrence of low levels of the 5-S-cysteinyl adducts in dopamine-rich brain areas, but not in cerebellum, is indirect evidence of in vivo autoxidation of DOPA, DOPAC, and dopamine. The products generated during catechol autoxidation, including quinones and reduced forms of oxygen, are known to be potentially cytotoxic.     

Studies on the distribution of calcitonin gene-related peptide-like and substance P-like immunoreactivities in rat hind limb muscles

Summary The tibialis anterior, extensor digitorum longus and soleus muscles in the rat were examined with respect to the presence of calcitonin gene-related peptide-like as well as substance P-like immunoreactivity. In some of the motor endplates in these muscles, identified by staining for acetylcholinesterase activity, calcitonin gene-related peptide-like immunoreactivity was detected, but in others it was not. Calcitonin gene-related peptide-like immunoreactivity was found to coexist with substance-P-like immunoreactivity in nerve fibres located outside and inside the capsule of the muscle spindles, as well as in nerve fibres located in nerve fascicles. These fibres presumably represent sensory nerve fibres. Calcitonin gene-related peptide-like immunoreactivity, but not substance P-like immunoreactivity, was also detected, in cap-like structures located on the surface of the intrafusal muscle fibres in the polar regions of the spindles, structures which are likely to correspond to motor plate endings. The observations suggest that calcitonin gene-related peptide is heterogeneously present in the endplates of rat hind limb muscles, and gives for the first time immunohistochemical evidence for the presence of calcitonin gene-related peptide and substance P in the innervation of muscle spindles.     

Photophysics of the fluorescent Ca2+ indicator Fura-2.

The photophysics of the complex forming reaction of Ca2+ and Fura-2 are investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with BAPTA as Ca2+ buffer: k01 = 1.2 x 10(9)s-1, k21 = 1.0 x 10(11) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 2.2 x 10(7) s-1, and with EGTA as Ca2+ buffer: k01 = 1.4 x 10(9) s-1, k21 = 5.0 x 10(10) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 3.2 x 10(7) s-1. k01 and k02 denote the respective deactivation rate constants of the Ca2+ free and bound forms of Fura-2 in the excited state. k21 represents the second-order rate constant of binding of Ca2+ and Fura-2 in the excited state, whereas k12 is the first-order rate constant of dissociation of the excited Ca2+:Fura-2 complex. The ionic strength of the solution was shown not to influence the recovered values of the rate constants. From the estimated values of k12 and k21, the dissociation constant K*d in the excited state was calculated. It was found that in EGTA Ca2+ buffer pK*d (3.2) is smaller than pKd (6.9) and that there is negligible interference of the excited-state reaction with the determination of Kd and [Ca2+] from fluorimetric titration curves. Hence, Fura-2 can be safely used as an Ca2+ indicator. From the obtained fluorescence decay parameters and the steady-state excitation spectra, the species-associated excitation spectra of the Ca2+ free and bound forms of Fura-2 were calculated at intermediate Ca2+ concentrations.     

Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin.

Furin, a subtilisin-like eukaryotic endoprotease, is responsible for proteolytic cleavage of cellular and viral proteins transported via the constitutive secretory pathway. Cleavage occurs at the C-terminus of basic amino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golgi network (TGN), but also in clathrin-coated vesicles dispatched from the TGN, on the plasma membrane as an integral membrane protein and in the medium as an anchorless enzyme. When furin was vectorially expressed in normal rat kidney (NRK) cells it accumulated in the TGN similarly to the endogenous glycoprotein TGN38, often used as a TGN marker protein. The signals determining TGN targeting of furin were investigated by mutational analysis of the cytoplasmic tail of furin and by using the hemagglutinin (HA) of fowl plague virus, a protein with cell surface destination, as a reporter molecule, in which membrane anchor and cytoplasmic tail were replaced by the respective domains of furin. The membrane-spanning domain of furin grafted to HA does not localize the chimeric molecule to the TGN, whereas the cytoplasmic domain does. Results obtained on furin mutants with substitutions and deletions of amino acids in the cytoplasmic tail indicate that wild-type furin is concentrated in the TGN by a mechanism involving two independent targeting signals, which consist of the acidic peptide CPSDSEEDEG783 and the tetrapeptide YKGL765. The acidic signal in the cytoplasmic domain of a HA-furin chimera is necessary and sufficient to localize the reporter molecule to the TGN, whereas YKGL is a determinant for targeting to the endosomes. The data support the concept that the acidic signal, which is the dominant one, retains furin in the TGN, whereas the YKGL motif acts as a retrieval signal for furin that has escaped to the cell surface.     

Muscle glycogen recovery after exercise during glucose and fructose intake monitored by 13C-NMR

Van Den Bergh, Adrianus J., Sibrand Houtman, ArendHeerschap, Nancy J. Rehrer, Hendrikus J. Van Den Boogert, BerendOeseburg, and Maria T. E. Hopman. Muscle glycogen recovery afterexercise during glucose and fructose intake monitored by¹³C-NMR. J. Appl.Physiol. 81(4): 1495-1500, 1996.The purpose of this study was to examine muscle glycogen recovery with glucose feeding(GF) compared with fructose feeding (FF) during the first 8 h afterpartial glycogen depletion by using¹³C-nuclear magneticresonance (NMR) on a clinical 1.5-T NMR system. After measurement of the glycogen concentration of the vastus lateralis (VL) muscle in seven male subjects, glycogen stores of the VLwere depleted by bicycle exercise. During 8 h after completion ofexercise, subjects were orally given either GF or FF while the glycogencontent of the VL was monitored by¹³C-NMR spectroscopy every secondhour. The muscular glycogen concentration was expressed as a percentageof the glycogen concentration measured before exercise. The glycogenrecovery rate during GF (4.2 ± 0.2%/h) was significantly higher(P < 0.05) compared withvalues during FF (2.2 ± 0.3%/h). This study shows that1) muscle glycogen levels areperceptible by ¹³C-NMRspectroscopy at 1.5 T and 2) theglycogen restoration rate is higher after GF compared with after FF.

     

A histological and immunohistochemical study of the effects of N-acetyl cysteine on retinopathy of prematurity by modifying insulin-like growth factor-1

Retinopathy of prematurity (ROP) is a vasoproliferative disorder that occurs in premature infants and may lead to permanent visual impairment. We investigated both the possible protective role of N-acetyl cysteine (NAC) for preventing ROP and the role of IGF-1 in the disorder. Forty-five newborn rats were divided into three groups. Group 1 was raised in room air as controls. Group 2 was exposed to 60% oxygen for 14 days after birth, then transferred to room air. Group 3 was exposed to the same conditions as group 2, but received intraperitoneal injections of NAC on postnatal days 7–17. After 35 days, both eyes of all rats were processed for histology. Some sections were stained with hematoxylin and eosin to assess structural changes and other sections were immunostained to determine the location of IGF-1. Frozen sections also were prepared and stained for adenosine triphosphatase to detect retinal blood vessels. Compared to the controls, more blood vessels, many of which were abnormal, and increased IGF-1 expression were observed in group 2. In group 3, abnormal blood vessels and IGF-1 expression were less evident. NAC appeared to be an effective vascular-protective agent for ROP by decreasing IGF-1 expression.     

Glutamic acid 71 and aspartic acid 66 control the binding of the second calcium ion in porcine pancreatic phospholipase A2

In addition to the Ca2+ ion at the active site, porcine pancreatic phospholipase A2 (PLA) is known to bind a second calcium ion with a lower affinity at alkaline pH. The second calcium-binding site has been held responsible for effective interaction of phospholipase with organized lipid/water interfaces [van Dam-Mieras, M. C. E., Slotboom, A. J., Pieterson, W. A. and de Haas, G. H. (1975) Biochemistry 14, 5387-5394]. To study the identity of the acidic amino acid residues involved in liganding the second calcium ion in detail, we used site-directed mutagenesis to specifically alter the cDNA encoding porcine pancreatic phospholipase. Three mutant phospholipase species were constructed, each of which lacked one of the potentially important carboxylates: Asp66----Asn, Glu71----Asn and Glu92----Gln. The Gln92 mutant PLA displayed the same properties as native phospholipase indicating that Glu92 is not important for binding the second metal ion. However, Glu71 and, to a lesser extent, Asp66 are both directly involved in the low-affinity calcium binding.     

The role of Asp-49 and other conserved amino acids in phospholipases A2 and their importance for enzymatic activity

The role of aspartic acid-49 (Asp-49) in the active site of porcine pancreatic phospholipase A2 was studied by recombinant DNA techniques: two mutant proteins were constructed containing either glutamic acid (Glu) or lysine (Lys) at position 49. Enzymatic characterization indicated that the presence of Asp-49 is essential for effective hydrolysis of phospholipids. Conversion of Asp-49 to either Glu or Lys strongly reduces the binding of Ca2+ ions, in particular for the lysine mutant, but the affinity for substrate analogues is hardly affected. Extensive purification of naturally occurring Lys-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus yielded a protein that was nearly inactive. Inhibition studies showed that this residual activity was due to a small amount of contaminating enzyme and that the Lys-49 homologue itself has no enzymatic activity. Our results indicate that Asp-49 is essential for the catalytic action of phospholipase A2. The importance of Asp-49 was further evaluated by comparison of the primary sequences of 53 phospholipases A2 and phospholipase homologues showing that substitutions at position 49 are accompanied by structural variations of otherwise conserved residues. The occurrence of several nonconserved substitutions appeared to be a general characteristic of nonactive phospholipase A2 homologues.     

Changes of testicular cholesteryl ester hydrolase activity in experimentally cryptorchid rats

A simple and reliable method was developed to determine the neutral cholesteryl ester hydrolase (CEH) activity in rat testes, using cholesteryl-[1-14C]-oleate as substrate. The activity was due to a soluble enzyme present in the cytoplasm of predominantly Sertoli cells, which could be shown after depleting the testes of Leydig cells with ethane dimethyl sulphonate. This treatment also revealed that the loss of CEH activity in abdominal testes of experimentally cryptorchid rats takes place in the Sertoli cells. In prepubertal rats made unilaterally cryptorchid at birth, the CEH activity was significantly higher in the abdominal than in the scrotal testes at 16 days of age. This is earlier than any previously described biochemical change and coincides with, or may even precede, the earliest morphological changes which are accumulation of lipid droplets in the Sertoli cells. The testicular CEH activity then decreased to 30 days of age in the abdominal testes, whereas the activity increased in the contralateral, scrotal testes. When adult rats were made unilaterally cryptorchid for 24 h, the CEH activity decreased rapidly in the abdominal testes. These results suggest that a derangement in cholesteryl ester metabolism is an early event in the pathogenesis of testicular degeneration in cryptorchidism.