Influence of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, pravastatin, on corticosteroid metabolism in patients with heterozygous familial hypercholesterolemia.

The present study examined whether hypolipidemic therapy with a potent 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, pravastatin, influences corticosteroid metabolism in patients with heterozygous familial hypercholesterolemia (FH). Urinary excretion of tetrahydrocortisone, tetrahydrocortisol, 6 beta-hydroxycortisol and free cortisol were determined in 22 patients with heterozygous FH before and after pravastatin administration (10 mg/day for 2 months). Pravastatin induced a statistically significant decrease in serum total cholesterol in patients with heterozygous FH from 6.9 +/- 0.1 to 5.9 +/- 0.1 mmol/l (p less than 0.05). No significant changes were seen in the urinary tetrahydrocortisone, tetrahydrocortisol and free cortisol levels before and after pravastatin therapy. Urinary excretion of 6 beta-hydroxycortisol was significantly (p less than 0.05) increased after pravastatin administration. These results suggest that the hypolipidemic effect of pravastatin in patients with heterozygous FH does not influence the corticosteroid metabolism. The increase in urinary 6 beta-hydroxycortisol may be caused by pravastatin-induced hepatic microsomal 6 beta-hydroxylase induction.     

Fructose transporter in human spermatozoa and small intestine is GLUT5.

We recently reported that the glucose transporter isoform, GLUT5, is expressed on the brush border membrane of human small intestinal enterocytes (Davidson, N. O., Hausman, A. M. L., Ifkovits, C. A., Buse, J. B., Gould, G. W., Burant, C. F., and Bell, G. I. (1992) Am. J. Physiol. 262, C795-C800). To define its role in sugar transport, human GLUT5 was expressed in Xenopus oocytes and its substrate specificity and kinetic properties determined. GLUT5 exhibits selectivity for fructose transport, as determined by inhibition studies, with a Km of 6 mM. In addition, fructose transport by GLUT5 is not inhibited by cytochalasin B, a competitive inhibitor of facilitative glucose transporters. RNA and protein blotting studies showed the presence of high levels of GLUT5 mRNA and protein in human testis and spermatozoa, and immunocytochemical studies localize GLUT5 to the plasma membrane of mature spermatids and spermatozoa. The biochemical properties and tissue distribution of GLUT5 are consistent with a physiological role for this protein as a fructose transporter.     

Genomic factors responsible for abnormal morphology of pollen tubes in the interspecific cross Nicotiana tabacum ×N. rustica

Nicotiana tabacum shows unilateral pollen-pistil incongruity with N. rustica. If N. tabacum is pollinated with N. rustica, growth of the pollen tube is arrested in the middle of the style, and abundant callose deposition, tube swelling and tube winding are observed. An attempt was made to clarify the genomic factors responsible for this pollen-pistil incongruity. N. tabacum was pollinated with N. paniculata or N. undulata, progenitors of amphidiploid N. rustica. When pollinated with N. undulata, growth of the pollen tube was arrested in the middle of the style and showed abnormal morphology similar to that with N. rustica, but when pollinated with N. paniculata the pollen tube reached near the base of the style and was almost normal in appearance. These observations suggest that the factors responsible for the pollen tube abnormality of N. rustica are derived from the N. undulata genome.We also used N. sylvestris, N. glutinosa and N. otophora as pistilar parents and N. rustica or its progenitors as pollen parents to examine the genomic factors of the pistilate parents. The pollen tube features of these three species in the pistils of N. sylvestris were similar to those in the pistil of N. tabacum. Received: 25 October 1999 / Revision accepted: 2 February 2000     

Cytokine-induced cell death in immortalized Schwann cells: roles of nitric oxide and cyclic AMP

Tumor necrosis factor-alpha and interferon-gamma are pleiotropic cytokines that regulate Schwann cell responses during injury and inflammatory demyelination. We have previously shown that cyclic AMP (cAMP)-elevating agents decrease the demyelination and Wallerian degeneration in experimental allergic neuritis. In this study, we examined the role of cAMP in cytokine-mediated signaling in a spontaneously immortal Schwann cell clone (iSC). We found that tumor necrosis factor-alpha and interferon-gamma exert synergistic inhibitory action on Schwann cell viability via the production of nitric oxide (NO) and ceramide (cer). Furthermore, we found that: (i) NO synthase inhibitors attenuate the cytokine-induced cer accumulation and cell death indicating that NO acts upstream of cer; and (ii) cytokine-induced cell death is decreased in iSCs pretreated continuously for 48-72 h with forskolin, an activator of adenylate cyclase. Although forskolin modulates the phosphorylation of ERKs and Akt, it decreases the susceptibility of iSC to cytokines via a separate mechanism operating after NO induction and before cer accumulation. We propose that the protective effect of cAMP-elevating agents in experimental allergic neuritis may be mediated in part via modulation of Schwann cell responses to cytokines.     

Phylogenetic study of the oxytocin-like immunoreactive system in invertebrates

1. A phylogenetic study of oxytocin (OXT)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Oncidium verrucosum, Limax marginatus and Meretrix lamarckii of the Mollusca; and Baratha brassica of the Arthropoda. 3. No immunoreactive cells were found in Bipalium sp. of the Platyhelminthes; Pomacea canaliculata, Aplysia kurodai, Bradybaena similaris and Achatina fulica of the Mollusca; and Gnorimosphaeroma rayi, Procambarus clarkii, Hemigrapsus sanguineus, Helice tridens and Gryllus bimaculatus of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 4. These results demonstrate that an OXT-immunoreactive substance is widely present not only in vertebrates but also in invertebrates. 5. OXT seems to have been introduced into these invertebrates at an early stage of their phylogenetic history.     

Simple But Efficacious Enrichment of Integral Membrane Proteins and Their Interactions for In-Depth Membrane Proteomics

Membrane proteins play essential roles in various cellular processes, such as nutrient transport, bioenergetic processes, cell adhesion, and signal transduction. Proteomics is one of the key approaches to exploring membrane proteins comprehensively. Bottom–up proteomics using LC–MS/MS has been widely used in membrane proteomics. However, the low abundance and hydrophobic features of membrane proteins, especially integral membrane proteins, make it difficult to handle the proteins and are the bottleneck for identification by LC–MS/MS. Herein, to improve the identification and quantification of membrane proteins, we have stepwisely evaluated methods of membrane enrichment for the sample preparation. The enrichment methods of membranes consisted of precipitation by ultracentrifugation and treatment by urea or alkaline solutions. The best enrichment method in the study, washing with urea after isolation of the membranes, resulted in the identification of almost twice as many membrane proteins compared with samples without the enrichment. Notably, the method significantly enhances the identified numbers of multispanning transmembrane proteins, such as solute carrier transporters, ABC transporters, and G-protein–coupled receptors, by almost sixfold. Using this method, we revealed the profiles of amino acid transport systems with the validation by functional assays and found more protein–protein interactions, including membrane protein complexes and clusters. Our protocol uses standard procedures in biochemistry, but the method was efficient for the in-depth analysis of membrane proteome in a wide range of samples.     

Amyloid β-Mediated Zn2+ Influx into Dentate Granule Cells Transiently Induces a Short-Term Cognitive Deficit

We examined an idea that short-term cognition is transiently affected by a state of confusion in Zn²⁺ transport system due to a local increase in amyloid-β (Aβ) concentration. A single injection of Aβ (25 pmol) into the dentate gyrus affected dentate gyrus long-term potentiation (LTP) 1 h after the injection, but not 4 h after the injection. Simultaneously, 1-h memory of object recognition was affected when the training was performed 1 h after the injection, but not 4 h after the injection. Aβ-mediated impairments of LTP and memory were rescued in the presence of zinc chelators, suggesting that Zn²⁺ is involved in Aβ action. When Aβ was injected into the dentate gyrus, intracellular Zn²⁺ levels were increased only in the injected area in the dentate gyrus, suggesting that Aβ induces the influx of Zn²⁺ into cells in the injected area. When Aβ was added to hippocampal slices, Aβ did not increase intracellular Zn²⁺ levels in the dentate granule cell layer in ACSF without Zn²⁺, but in ACSF containing Zn²⁺. The increase in intracellular Zn²⁺ levels was inhibited in the presence of CaEDTA, an extracellular zinc chelator, but not in the presence of CNQX, an AMPA receptor antagonist. The present study indicates that Aβ-mediated Zn²⁺ influx into dentate granule cells, which may occur without AMPA receptor activation, transiently induces a short-term cognitive deficit. Extracellular Zn²⁺ may play a key role for transiently Aβ-induced cognition deficits.     

Anterograde Axonal Transport of Peptidylglycine α-Amidating Monooxygenase in Rat Sciatic Nerves

Axonal transport of peptidylglycine alpha-amidating monooxygenase (PAM) activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and reached a plateau between 48 and 72 h and then decreased. The flow rate was 100 mm/day, and the molecular mass of the active entity was 70 kDa, which was determined by gel filtration. In contrast, there was no evidence for significant retrograde axonal transport. Anterograde axonal transport of immunoreactive cholecystokinin, a carboxy-terminal-amidated putative neuropeptide, was also found. These results suggest that PAM is transported by a rapid axonal flow and may play a role as a processing enzyme during transport or in the terminals of rat sciatic nerves.     

Production, crystallization, and preliminary X-ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1-47) of troponin I.

Troponin is a ternary protein complex consisting of subunits TnC. TnI, and TnT, and plays a key role in calcium regulation of the skeletal and cardiac muscle contraction. In the present study, a partial complex (CI47) was prepared from Escherichia coli-expressed rabbit skeletal muscle TnC and fragment 1-47 of TnI, which is obtained by chemical cleavage of an E. coli-expressed mutant of rabbit skeletal muscle TnI. Within the ternary troponin complex, CI47 is thought to form a core that is resistant to proteolytic digestion, and the interaction within CI47 likely maintains the integrity of the troponin complex. Complex CI47 was crystallized in the presence of sodium citrate. The addition of trehalose improved the diffraction pattern of the crystals substantially. The crystal lattice belongs to the space group P3(1)(2)21, with unit cell dimensions a = b = 48.2 A, c = 162 A. The asymmetric unit presumably contains one CI47 complex. Soaking with p-chloromercuribenzenesulfonate (PCMBS) resulted in loss of isomorphism, but enhanced the quality of the crystals. The crystals diffracted up to 2.3 A resolution, with completeness of 91% and R(merge) = 6.4%. The crystals of PCMBS-derivative should be suitable for X-ray studies using the multiple-wavelength anomalous diffraction technique. This is the first step for elucidating the structure of the full troponin complex.