Cloning and Characterization of the cDNA Encoding a Novel Brain-Specific 14-kDa Protein

A new acidic protein with a molecular weight of 14,000 was purified from rat brain, in which it was specifically expressed, and partially sequenced by protein sequencing. On the basis of results obtained from the amino acid sequences, mixed oligonucleotides were synthesized and used as probes to clone a cDNA from a rat brain cDNA library. The cloned cDNA provided the full-length sequence of the 14-kDa protein. Northern blot hybridization using total RNA from several tissues of the rat provided evidence that the 14-kDa protein was expressed specifically in rat brain. Transfection of this cDNA into mammalian cells resulted in expression of the 14-kDa protein. The amino acid sequence predicted from the cDNA of the rat brain 14-kDa protein contained 137 amino acid residues. A hydropathy profile revealed a hydrophobic domain (amino acids 60-80) flanked by highly hydrophilic stretches on both sides. Whereas the N-terminal region of the 14-kDa protein contained four repeating motifs, EKTKEGV, the C-terminal domain was rich in glutamic acid and proline. A computer search of the amino acid sequence of the 14-kDa protein indicated no homology to any other protein reported so far.     

Isolation of NPY-25 (neuropeptide Y[12-36]), a potent inhibitor of calmodulin, from porcine brain

In the present purification of low molecular weight fractions (Mr: 2000-4000) containing basic peptides, twenty nmol of novel calmodulin binding peptide, possessing a potent affinity for calmodulin, was isolated from 18 kg of porcine brain. By analysis with gas phase sequencer, the sequence was determined to be APAEDLARYYSALRHYINLITRQRY. Carboxy terminus of the peptide was determined to be Tyr-NH2. The peptide was a carboxy terminal pentacosanepeptide of neuropeptide Y and was termed NPY-25. NPY-25 competitively inhibited the activation of cAMP-phosphodiesterase through CaM binding in a Ca++ dependent fashion, but did not inhibit the basal activity of cAMP phosphodiesterase. NPY-25 elicited a more potent activity than did neuropeptide Y. IC50 values of NPY-25 and Neuropeptide Y were 0.06 microM and 0.54 microM respectively.     

Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice

We recently reported that mice deficient in the programmed cell death-1 (PD-1) immunoinhibitory coreceptor develop autoimmune dilated cardiomyopathy (DCM), with production of high-titer autoantibodies against a heart-specific, 30-kDa protein. In this study, we purified the 30-kDa protein from heart extract and identified it as cardiac troponin I (cTnI), encoded by a gene in which mutations can cause familial hypertrophic cardiomyopathy (HCM). Administration of monoclonal antibodies to cTnI induced dilatation and dysfunction of hearts in wild-type mice. Monoclonal antibodies to cTnI stained the surface of cardiomyocytes and augmented the voltage-dependent L-type Ca2+ current of normal cardiomyocytes. These findings suggest that antibodies to cTnI induce heart dysfunction and dilatation by chronic stimulation of Ca2+ influx in cardiomyocytes.     

Sexual modification of female spinach seeds (Spinacia oleracea L.) by irradiation with ion particles

The female seeds of a spinach plant (Spinacia orelacea L.) were exposed to He (12.5 MeV/n) and C (18.3 MeV/n) ions in order to investigate the effects of ion particles on sex expression. He ions did not affect germination rates or flowering at doses up to 50 Gy. C ions did not affect germination rates or flowering at doses up to 15 Gy, but a dose of 25 Gy resulted in many plants with morphological aberrations. When unexposed female plants were grown without cross-fertilization for 10 weeks after sowing, 5.6-14.3% of the plants produced anthers from female flowers. These sex-modified plants could self-pollinate and form seeds, which expressed only female organs. Conversely, gynomonoecious plants were induced from these female seeds by exposure to He ions (5-50 Gy) and C ions (5-25 Gy) without any difference in the rates of flowered progeny. Moreover, andromonoecious plants were induced from female seeds by exposure to He ions at 50 Gy. These results suggest that the sex of a spinach plant is expressed as a flexible phenotype, converging from female to gyno- and andromonoecy after exposure to ion particles.     

A gene for a Class II DNA photolyase from<Emphasis Type="Italic"> Oryza sativa</Emphasis>: cloning of the cDNA by dilution-amplification

Ultraviolet radiation induces the formation of two classes of photoproducts in DNA-the cyclobutane pyrimidine dimer (CPD) and the pyrimidine [6-4] pyrimidone photoproduct (6-4 product). Many organisms produce enzymes, termed photolyases, which specifically bind to these lesions and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage. These photolyases are specific for either CPDs or 6-4 products. Two classes of photolyases (class I and class II) repair CPDs. A gene that encodes a protein with class II CPD photolyase activity in vitro has been cloned from several plants including Arabidopsis thaliana, Cucumis sativus and Chlamydomonas reinhardtii. We report here the isolation of a homolog of this gene from rice (Oryza sativa), which was cloned on the basis of sequence similarity and PCR-based dilution-amplification. The cDNA comprises a very GC-rich (75%) 5; region, while the 3; portion has a GC content of 50%. This gene encodes a protein with CPD photolyase activity when expressed in E. coli. The CPD photolyase gene encodes at least two types of mRNA, formed by alternative splicing of exon 5. One of the mRNAs encodes an ORF for 506 amino acid residues, while the other is predicted to code for 364 amino acid residues. The two RNAs occur in about equal amounts in O. sativa cells.     

Design and synthesis of novel pyrimido[4,5-b]azepine derivatives as HER2/EGFR dual inhibitors

A novel 7,6 fused bicyclic scaffold, pyrimido[4,5-b]azepine was designed to fit into the ATP binding site of the HER2/EGFR proteins. The synthesis of this scaffold was accomplished by an intramolecular Claisen-type condensation. As the results of optimization lead us to 4-anilino and 6-functional groups, we discovered 6-substituted amide derivative 19b, which has a 1-benzothiophen-4-yloxy group attached to the 4-anilino group. An X-ray co-crystal structure of 19b with EGFR demonstrated that the N-1 and N-3 nitrogens of the pyrimido[4,5-b]azepine scaffold make hydrogen-bonding interactions with the main chain NH of Met793 and the side chain of Thr854 via a water-mediated hydrogen bond network, respectively. In addition, the NH proton at the 9-position makes an additional hydrogen bond with the carbonyl group of Met793, as we expected. Compound 19b revealed potent HER2/EGFR kinase (IC₅₀: 24/36 nM) and BT474 cell growth (GI₅₀: 18 nM) inhibitory activities based on its pseudo-irreversible (PI) profile.     

Biochemical investigations of the nuclear proteins during the transition of root meristems from quiescent to proliferating state in Pisum sativum

Abstract

The electrophoretic analysis of nuclear proteins extracted from root meristems at different times of germination puts in evidence the variations of content of specific proteins. Several nuclear proteins are phosphorylated by endogenous protein kinase and often the maximum rate of phosphorylation it has been observed in proteins present in the nucleus at low concentrations. Moreover also the phosphorylation rate of specific proteins changes at different times of germination. It is interesting the fact that both variations of concentration and phosphorylation in nuclear proteins occurr at the time when root meristems leave the quiescence to enter a proliferating state. We suggest that these variations play a role in this physiological event.     

Phospholipase C-delta1 and -delta3 are essential in the trophoblast for placental development

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in phosphoinositide turnover and is involved in a variety of physiological functions. We analyzed PLCdelta1 knockout mice and found that PLCdelta1 is required for the maintenance of skin homeostasis. However, there were no remarkable abnormalities except hair loss and runting in PLCdelta1 knockout mice, even though PLCdelta1 is broadly distributed. Here, we report that mice lacking both PLCdelta1 and PLCdelta3 died at embryonic day 11.5 (E11.5) to E13.5. PLCdelta1/PLCdelta3 double-knockout mice exhibited severe disruption of the normal labyrinth architecture in the placenta and decreased placental vascularization, as well as abnormal proliferation and apoptosis of trophoblasts in the labyrinth area. Furthermore, PLCdelta1/PLCdelta3 double-knockout embryos supplied with a normal placenta by the tetraploid aggregation method survived beyond E14.5, clearly indicating that the embryonic lethality is caused by a defect in trophoblasts. On the basis of these results, we conclude that PLCdelta1 and PLCdelta3 are essential in trophoblasts for placental development.