Changes in Levels of Purine and Pyrimidine Nucleotides During Acute Hypoxia and Recovery in Neonatal Rat Brain

Neonatal rat brains were examined for changes in levels of ATP, ADP, AMP, cyclic AMP, GTP, GDP, UTP, UDP, UMP, and CTP during exposure to 100% nitrogen for 20 min and subsequent recovery in air. During hypoxia, ATP, GTP, UTP, and CTP levels and the GTP/GDP ratio decreased to 38, 50, 26, 21, and 21%, respectively, of control levels. No significant change in cyclic AMP level was observed. The decrease in the total uridine nucleotide pool during hypoxia was markedly greater (to 53% of control levels) than that in the total adenine nucleotide pool (to 92% of control levels). During recovery, ATP and GTP levels were rapidly and almost completely restored. On the other hand, CTP levels returned slowly to control values after a 2-h recovery period. Restoration of the UTP level was slow and incomplete (87% of the control value even after a 3-h recovery period). The GTP/GDP ratio also did not return to normal. These data suggest that hypoxic insult to the neonate may have an effect on the synthesis of nucleotidyl sugars, phospholipids, and proteins in the brain, resulting in significant problems with developmental processes of the brain. The present study also showed that the delayed restorations of the UTP level and the GTP/GDP ratio were not seen in the brains of adult rats subjected to acute severe hypoxic insult.     

Purification and Characterization of Benzonitrilases from Arthrobacter sp. Strain J-1

We found two kinds of benzonitrilases, designated benzonitrilases A and B, in a cell extract of Arthrobacter sp. strain J-1 grown on benzonitrile as a sole carbon and nitrogen source. Benzonitrilases A and B were purified approximately 409-fold and 38-fold, respectively. Purified benzonitrilase A appeared to be homogeneous according to the criteria of polyacrylamide gel electrophoresis. Both the enzymes hydrolyzed benzonitrile to benzoic acid and ammonia without forming benzamide as an intermediate. The molecular weights of benzonitrilases A and B were found to be 30,000 and 23,000, respectively. The subunit molecular weight of benzonitrilase A was the same as its molecular weight. The isoelectric points of benzonitrilases A and B were 4.95 and 4.80, respectively. The optimum temperature and pH, respectively, for benzonitrilase A were 40°C and 8.5, and those for benzonitrilase B were 30°C and 7.5. The K_m values for benzonitrilases A and B were 6.7 mM and 4.5 mM, respectively. Both the enzymes degraded p-tolunitrile, 4-cyanopyridine, and p-chlorobenzonitrile, but they did not attack aliphatic nitriles or amides. Both the enzymes were inhibited by thiol reagents.     

Difference between N-acetylgalactosamine 4-sulfate 6-O-sulfotransferases from human serum and squid cartilage in specificity toward the terminal and interior portion of chondroitin sulfate

In the preceding paper (Inoue, H., Otsu, K., Yoneda, M., Kimata, K., Suzuki, S., and Nakanishi, Y. (1986) J. Biol. Chem. 261, 4460-4469), we reported the purification from human serum of an N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase fraction which was able to transfer sulfate predominantly to position 6 of the nonreducing terminal N-acetylgalactosamine 4-sulfate unit of chondroitin sulfate. We now show that the activity toward the terminal was co-purified with a minor activity toward the interior counterpart by sequential chromatography on heparin-Sepharose CL-6B, Matrex Blue B, hydroxyapatite, and Sephacryl S-300, and that the two activities were equally heatlabile. The enzyme purified 5000-fold from human serum was devoid of the sulfotransferase activities toward chondroitin, heparan sulfate, and keratan sulfate, but showed a strong terminal sulfotransferase activity toward dermatan sulfate (pig skin); over 97% of the sulfate residues incorporated were at position 6 of the nonreducing N-acetylgalactosamine 4,6-bissulfate end groups linked to the L-iduronic acid group. Although the enzyme introduces sulfate predominantly into the nonreducing terminal of chondroitin sulfate at physiological pH (approximately equal to 7.0) and Ca2+ concentration (approximately 2-3 mM), the activity toward the interior portion relative to that toward the terminal was increased by either lowering pH or elevating Ca2+ concentration, perhaps owing to changes in the conformation or ionic state of the acceptor molecule. Comparison between the human serum enzyme and the N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (formerly designated "E6-sulfotransferase") from squid cartilage indicated that the latter is distinct from the former in introducing sulfate predominantly into the interior portion of chondroitin sulfate. It appears that the role of the squid sulfotransferase is to synthesize so-called chondroitin sulfate E where over 50% of the interior hexosamine units are 4,6-bis-sulfated.     

A terminal 6-sulfotransferase catalyzing a synthesis of N-acetylgalactosamine 4,6-bissulfate residue at the nonreducing terminal position of chondroitin sulfate

A soluble enzyme from quail oviduct which incorporates sulfate into position 6 of the nonreducing N-acetylgalactosamine 4-sulfate end group of chondroitin sulfate has been purified. This enzyme (termed "terminal 6-sulfotransferase") was partially separated from a 6-sulfotransferase present in the same tissue which catalyzes the incorporation of sulfate into interior portion of unsulfated chondroitin. The basic requirements for the terminal 6-sulfotransferase reaction were shown to be 3'-phosphoadenylyl sulfate (donor) and chondroitin 4-sulfate (acceptor). The substitution of unsulfated chondroitin (prepared from squid skin) for chondroitin 4-sulfate resulted in a total loss of activity. These results suggest that the organization of the proteoglycan-synthesizing apparatus may well involve hitherto unrecognized mechanisms for the sulfation of chondroitin chains.     

Increase in Striatal [3H]Muscimol Binding Following Intrastriatal Injection of Kainic Acid: A Denervation Supersensitivity Phenomenon

The effect of intrastriatal microinjection of kainic acid (KA) on specific binding of [³H]muscimol to the particulate fractions obtained from corpus striatum (CS), globus pallidus (GP), substantia nigra (SN), and cerebral cortex (CC) was examined. Seven days after the unilateral intrastriatal microinjection of KA, the amount of specifically bound [³H]muscimol was significantly increased at the injected site, whereas no significant alteration of [³H]muscimol binding was found in GP, SN, or CC. Scatchard analysis of striatal binding revealed that microinjection of KA significantly increased the affinity (K_D) of GABA receptors on the injected (lesioned) side of the CS without affecting the total number of binding sites (B_max) therein. This significant increase in [³H]muscimol binding, however, was eliminated by pretreating particulate fractions from the CS with Triton X-100, a non-ionic detergent. No statistically significant difference in amounts of [³H]muscimol binding was detected when the preparations from the KA-treated and non-treated CS were preincubated with 0.05% Triton X-100, respectively. Scatchard analysis using CS preparations treated with 0.05% Triton X-100 revealed that the affinity of the GABA receptor was increased by treatment with Triton X-100, while the total number of binding sites (B_max) was unchanged by this treatment. These results suggest that neuronal degeneration produced by KA in vivo and pretreatment of particulate preparations with Triton X-100 in vitro may increase the amount of specifically bound [³H]muscimol to CS preparations by a similar molecular mechanism.     

Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase.

Affinity-purified bovine brain phosphatidylinositol 3-kinase (PI3-kinase) contains two major proteins of 85 and 110 kd. Amino acid sequence analysis and cDNA cloning reveals two related 85 kd proteins (p85 alpha and p85 beta), which both contain one SH3 and two SH2 regions (src homology regions). When expressed, these 85 kd proteins bind to and are substrates for tyrosine-phosphorylated receptor kinases and the polyoma virus middle-T antigen/pp60c-src complex, but lack PI3-kinase activity. However, an antiserum raised against p85 beta immunoprecipitates PI3-kinase activity. The active PI3-kinase complex containing p85 alpha or p85 beta and the 110 kd protein binds to PDGF but not EGF receptors. p85 alpha and p85 beta may mediate specific PI3-kinase interactions with a subset of tyrosine kinases.     

Immunocytochemical localization of creatine kinase M in canine myocardial cells: most creatine kinase M is distributed in the A-band

The localization of creatine kinase (CK) M in canine myocardium was immunocytochemically studied by a direct immunoperoxidase method. Specific antiserum against CK-M was produced in rabbits immunized with canine CK-MM. An anti-CK-M Fab'-horseradish peroxidase conjugate was prepared by the maleimide method. Frozen sections prepared from fixed canine myocardium were stained with the conjugate and observed by light and electron microscopy. In light microscopy of longitudinal sections, CK-M showed a cross-striated pattern consisting of distinct broad and narrow brown bands. Immunoelectron microscopy revealed that the regions of the broad and narrow brown bands corresponded to the A-band and the Z-line, respectively. Most CK-M in the A-band was associated with the thick fibers, and a small amount of CK-M was found in the M-line. These findings suggest that ATP regeneration from the ADP produced by myosin ATPase is related to the participation of this CK associated with the thick fibers rather than that of the M-line-bound CK. Creatine kinase M was also found in the sarcolemmal membrane, the membranes of the sarcoplasmic reticulum, and the mitochondrial outer and inner membranes. This report provides new information for understanding the physiological role of the phosphorylcreatine shuttle in the myocardial energy transport system.     

Immunohistochemical demonstration of GABAB receptors in the rat gastrointestinal tract

Immunohistochemical localization of GABA_B-receptors was demonstrated in the rat gastrointestinal tract using a monoclonal antibody (GB-1) raised against the purified GABA_B-receptor. Immunoreactive staining for GABA_B-receptors was found in some populations of endocrine, muscular and neuronal components in the stomach and gut wall. Positive mucosal epithelial, probably endocrine, cells were distributed throughout the stomach and intestine. Double immunostaining indicated that such positive cells for GABA_B-receptors often co-possessed serotonin in the small intestine but not in the gastric body. In the muscular layer of the digestive canal, positive staining was seen as dotty granules punctuated on the surface of muscle fibers. In the enteric nervous system, positive neuronal somata were found in both submucosal and myenteric ganglia throught the entire canal extending from the stomach to the rectum. This is the first report to visualize the cellular localization of GABA_B-receptors in the gastrointestinal system of the rat, and should provide a fundamental basis for future studies on gastrointestinal functions regulated by GABA_B-receptors. Special issue dedicated to Dr. Kinya Kuriyama.     

bcl-2-specific siRNAs restore gemcitabine sensitivity in human pancreatic cancer cells

Gemcitabine has been shown to ameliorate disease related symptoms and to prolong overall survival in pancreatic cancer.Yet, resistance to Gemcitabine is commonly observed in this tumour entity and has been linked to increased expression of anti-apoptotic bcl-2. We therefore investigated if and to what extend silencing of bcl-2 by specific siRNAs (siBCL2) might enhance Gemcitabine effects in human pancreatic carcinoma cells. siBCL2 was transfected into the pancreatic cancer cell line YAP C alone and 72 hrs before co-incubation with different concentrations of Gemcitabine. Total protein and RNA were extracted for Western-blot analysis and quantitative polymerase chain reaction. Pancreatic cancer xenografts in male nude mice were treated intraperitoneally with siBCL2 alone, Gemcitabine and control siRNA or Gemcitabine and siBCL2 for 21 days. Combination of both methods lead to a synergistic induction of apoptosis at otherwise ineffective concentrations of Gemcitabine. Tumour growth suppression was also potentiated by the combined treatment with siBCL2 and Gemcitabine in vivo and lead to increased TUNEL positivity. In contrast, non-transformed human foreskin fibroblasts showed only minor responses to this treatment. Our results demonstrate that siRNA-mediated silencing of anti-apoptotic bcl-2 enhances chemotherapy sensitivity in human pancreatic cancer cells in vitro and might lead to improved therapy responses in advanced stages of this disease.