Characterization of Microtubule-Associated Protein 2 from Mouse Brain and Its Localization in the Cerebellar Cortex

Microtubule-associated protein (MAP) 2 was purified from the microtubule fraction of mouse brain by heat treatment and BioGel A-5m gel filtration. The purified preparation showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis using both a gradient gel (3.75-12.5%) and a low-percentage gel (5%), a finding indicating that MAP2B was absent under the conditions used. Amino acid analysis revealed that mouse MAP2 was an acidic protein with an isoelectric point (pI 4.5) and amino acid composition similar to those of porcine brain MAP2. Immunoblot analysis indicated that the antigens that reacted with MAP2 antiserum were present in large quantities in mouse brain. However, we also found a weak reaction in various tissues other than brain, and the major antigens involved were recognized to be common molecular species with the same molecular mass, 162 and 170 kilodaltons. Using antiserum against mouse brain MAP2, the developmental localization patterns of MAP2 in the mouse cerebellar cortex were studied by immunohistochemistry. MAP2 was mainly localized in the neuronal cells throughout development, with the expression in Purkinje cell dendrites being especially remarkable in the growth of arborization from postnatal day 3 to day 20. At the mature stage, the reaction was strong in the dendritic tree but very weak in the proximal dendrites and cell bodies.     

Leaf ultrastructure of C4 species possessing different Kranz anatomical types in the cyperaceae

The leaf ultrastructure of NADP-malic enzyme type C₄ species possessing different anatomical features in the Cyperaceae was examined: types were the Rhynchosporoid type, a normal Kranz type in which mesophyll cells are adjacent to Kranz cells, and Fimbristyloid and Chlorocyperoid types, unusual Kranz types in which nonchlorophyllous mestome sheath intervenes between the two types of green cells. They show structural characteristics basically similar to the NADP-malic enzyme group of C₄ grasses, that is, centrifugally located chloroplasts with reduced grana and no increase of mitochondrial frequency in the Kranz cells. However, the Kranz cell chloroplasts of the Fimbristyloid and Chlorocyperoid types exhibit convoluted thylakoid systems and a trend of extensive development of peripheral reticulum, although those of the Rhynchosporoid type do not possess such particular membrane systems. The suberized lamella, probably a barrier for CO₂ diffusion, is present in the Kranz cell walls of the Rhynchosporoid type and in the mestome sheath cell walls of the other two types, and tightly surrounds the Kranz cells (sheaths) that are the sites of the decarboxylation of C₄ acids. These ultrastructural features are discussed in relation to C₄ photosynthetic function.     

Protective effect of cerulein on memory impairment induced by protein synthesis inhibitors in rats.

Previous studies have demonstrated that NMDA receptor antagonists and protein kinase C inhibitors induced marked memory impairment in rats, but that peripherally administered cerulein (CER) prevented these effects. In the present study, the effect of subcutaneously administered CER on amnesia induced by protein synthesis inhibitors was examined in passive and active avoidance responses and in the Morris water maze test. Intraperitoneal injection of the inhibitors produced marked memory impairment, but the effect was abolished by combined administration with CER. The effective dose of subcutaneously injected CER was, on a molar basis, three thousand- and six thousandfold less than the dose of anisomycin, and two hundred eighty- and three thousandfold less than the dose of puromycin in the passive and active avoidance response experiments, respectively. Similarly, in the Morris water maze test, behavioral disturbances produced by the protein synthesis inhibitors were abolished by CER. These results indicate the effectiveness of CER in preventing memory impairment induced by protein synthesis inhibitors.     

Effects of intracerebroventricular administration of opiate receptor antagonists on the suppressed pulsatile LH release during acute fasting in ovariectomized estradiol-treated rats.

The involvement of specific opiate receptors in the suppression of LH release during acute fasting in ovariectomized estradiol-treated rats was examined by intracerebroventricular (i.c.v.) administration of opiate receptor antagonists that exert a specificity directed mainly, although not absolutely, towards the delta-, kappa- or mu-opiate receptors. Fasting for 48 h significantly decreased mean plasma LH levels in estradiol-treated animals by increasing sensitivity to the negative feedback effect of estradiol. Injecting i.c.v. the mu-opiate receptor antagonist naloxone (10 or 100 nmol in 2 microliters of saline) blocked the inhibitory effect of fasting on pulsatile LH release and reinstated LH pulses. On the other hand, i.c.v. administration of the same dosages of a delta-opiate receptor antagonist ICI 174,864 or a kappa-opiate receptor antagonist WIN 44441-3 did not have any effect. These results suggest that the increased sensitivity of the LH-releasing mechanism to the negative feedback effect of estradiol during fasting involves the endogenous opioids mainly through the selective activation of the mu-opiate receptors.     

Requirement of c-kit for development of intestinal pacemaker system.

A discovery that the protooncogene encoding the receptor tyrosine kinase, c-kit, is allelic with the Dominant white spotting (W) locus establishes that c-kit plays a functional role in the development of three cell lineages, melanocyte, germ cell, and hematopoietic cell which are defective in W mutant mice. Recent analyses of c-kit expression in various tissues of mouse, however, have demonstrated that c-kit is expressed in more diverse tissues which are phenotypically normal in W mutant mice. Thus, whether or not c-kit expressed outside the three known cell lineages plays a functional role is one of the important questions needing answering in order to fully elucidate the role of c-kit in the development of the mouse. Here, we report that some of the cells in smooth muscle layers of developing intestine express c-kit. Blockade of its function for a few days postnatally by an antagonistic anti-c-kit monoclonal antibody (mAb) results in a severe anomaly of gut movement, which in BALB/c mice produces a lethal paralytic ileus. Physiological analysis indicates that the mechanisms required for the autonomic pacing of contraction in an isolated gut segment are defective in the anti-c-kit mAb-treated mice, W/Wv mice and even W/+ mice. These findings suggest that c-kit plays a crucial role in the development of a component of the pacemaker system that is required for the generation of autonomic gut motility.     

A homologous sequence between H+-ATPase (F0F1) and cation-transporting ATPases. Thr-285----Asp replacement in the beta subunit of Escherichia coli F1 changes its catalytic properties

A sequence of 10 amino acids (I-C-S-D-K-T-G-T-L-T) of ion motive ATPases such as Na+/K+-ATPase is similar to the sequence of the beta subunit of H+-ATPases, including that of Escherichia coli (I-T-S-T-K-T-G-S-I-T) (residues 282-291). The Asp (D) residue phosphorylated in ion motive ATPase corresponds to Thr (T) of the beta subunit. This substitution may be reasonable because there is no phosphoenzyme intermediate in the catalytic cycle of F1-ATPase. We replaced Thr-285 of the beta subunit by an Asp residue by in vitro mutagenesis and reconstituted the alpha beta gamma complex from the mutant (or wild-type) beta and wild-type alpha and gamma subunits. The uni- and multisite ATPase activities of the alpha beta gamma complex with mutant beta subunits were about 20 and 30% of those with the wild-type subunit. The rate of ATP binding (k1) of the mutant complex under uni-site conditions was about 10-fold less than that of the wild-type complex. These results suggest that Thr-285, or the region in its vicinity, is essential for normal catalysis of the H+-ATPase. The mutant complex could not form a phosphoenzyme under the conditions where the H+/K+-ATPase is phosphorylated, suggesting that another residue(s) may also be involved in formation of the intermediate in ion motive ATPase. The wild-type alpha beta gamma complex had slightly different kinetic properties from the wild-type F1, possibly because it did not contain the epsilon subunit.     

Purification and identification of [hydroxyprolyl3]bradykinin in ascitic fluid from a patient with gastric cancer

Kinins in the ascitic fluid from a patient with gastric cancer were purified by gel filtration and reversed-phase high-performance liquid chromatography (HPLC). Two fractions (fractions I and II) showed kinin activity. Fraction I did not correspond to either bradykinin or other known kinins, whereas fraction II corresponded to bradykinin. Fraction I contained 8 amino acid residues from bradykinin minus 1 proline plus 1 additional hydroxyproline. Sequence analysis of fraction I showed that the proline at the third amino acid residue of bradykinin was replaced by hydroxyproline. The retention time of fraction I on reversed-phase HPLC was exactly the same as that of synthetic [hydroxyprolyl3]bradykinin (Arg-Pro-Hyp-Gly-Phe-Ser-Pro-Phe-Arg) and was distinguishable from des-Pro3-bradykinin. Thus, these results demonstrate for the first time the presence of [hydroxyprolyl3]bradykinin in vivo. This is also the first report of the presence of bradykinin in human tumor ascites.