Vectorially oriented monolayers of the cytochrome c/cytochrome oxidase bimolecular complex

Vectorially oriented monolayers of yeast cytochrome c and its bimolecular complex with bovine heart cytochrome c oxidase have been formed by self-assembly from solution. Both quartz and Ge/Si multilayer substrates were chemical vapor deposited with an amine-terminated alkylsiloxane monolayer that was then reacted with a hetero-bifunctional cross-linking reagent, and the resulting maleimide endgroup surface then provided for covalent interactions with the naturally occurring single surface cysteine 102 of the yeast cytochrome c. The bimolecular complex was formed by further incubating these cytochrome c monolayers in detergent-solubilized cytochrome oxidase. The sequential formation of such monolayers and the vectorially oriented nature of the cytochrome oxidase was studied via meridional x-ray diffraction, which directly provided electron density profiles of the protein(s) along the axis normal to the substrate plane. The nature of these profiles is consistent with previous work performed on vectorially oriented monolayers of either cytochrome c or cytochrome oxidase alone. Furthermore, optical spectroscopy has indicated that the rate of binding of cytochrome oxidase to the cytochrome c monolayer is an order of magnitude faster than the binding of cytochrome oxidase to an amine-terminated surface that was meant to mimic the ring of lysine residues around the heme edge of cytochrome c, which are known to be involved in the binding of this protein to cytochrome oxidase.     

In vitro mutagenesis of the mitochondrial leucyl tRNA synthetase of Saccharomyces cerevisiae shows that the suppressor activity of the mutant proteins is related to the splicing function of the wild-type protein

The NAM2 gene of Saccharomyces cerevisiae encodes the mitochondrial leucyl tRNA synthetase (mLRS), which is necessary for the excision of the fourth intron of the mitochondrial cytb gene (bI4) and the fourth intron of the mitochondrial coxI gene (aI4), as well as for mitochondrial protein synthesis. Some dominant mutant alleles of the gene are able to suppress mutations that inactivate the bI4 maturase, which is essential for the excision of the introns aI4 and bI4. Here we report mutagenesis studies which focus on the splicing and suppressor functions of the protein. Small deletions in the C-terminal region of the protein preferentially reduce the splicing, but not the synthetase activity; and all the C-terminal deletions tested abolish the suppressor activity. Mutations which increase the volume of the residue at position 240 in the wild-type mLRS without introducing a charge, lead to a suppressor activity. The mutant 238C, which is located in the suppressor region, has a reduced synthetase activity and no detectable splicing activity. These data show that the splicing and suppressor functions are linked and that the suppressor activity of the mutant alleles results from a modification of the wild-type splicing activity.     

Impaired processing of proopiomelanocortin in corticotroph macroadenomas

The p53 mutations in the reported 243 lung cancers and our 59 cases were analyzed. The common base substitutions found in the lung cancer were G to T transversion (35%), C:G to T:A (26%) and A:T to G:C transitions (11%). Four types of the hot spot, 1) G to T transversion, 2) A to G transition in ApT site, 3) C to T transition in CpG site and 4) mix of the transversion and transition were identified. It is suggested that A to G transition at ApT site in the non-transcribed strand may be a new hot spot in the p53 gene in lung carcinoma. We also show that microscopic selection of cancer cells will facilitate the detection of p53 mutations in adenocarcinomas.     

Changes in myocardial A1 adenosine receptor and message levels during fetal development and postnatal maturation

Dysembryoplastic neuroepithelial tumor (DNT) is a recently described rare brain neoplasm with characteristic clinical and morphological features and favorable prognosis. We report here two cases of DNT. The first concerned a 12 years old girl who presented complex seizures preceded by acoustic aura (melodies). Computed tomography revealed a hypodense tumor measuring 2 x 2.5 cm in diameter, located paracortically in the left temporal lobe. The second tumor was removed from a 21-year-old man with partial complex seizures. Nine years earlier patient underwent neurosurgery with partial removal of the tumor The tumor's histopathologic diagnosis is unfortunately lacking. Computed and magnetic resonance imaging showed a mass occupying the cortex and paracortical areas of the anterior pole of the temporal lobe. Histologically, both tumors consisted of small, S-100 protein immunopositive oligodendrocyte-like cells (OLCs) arranged between synaptophysin- and, to a lesser degree, NFP-immunopositive axons (glioneuronal element). In the second case, an area of pilocytic astrocytoma-like appearance was also found, these cells were immunopositive for GFAP. The present study provides clinical, radiological and histological data, which may be helpful in differential diagnosis of this newly recognised brain tumor.     

A postmortem study of frontal cortical dopamine D1 receptors in schizophrenics, psychiatric controls, and normal controls

We tested the hypothesis that aberrant dopaminergic innervation in frontal and cingulate cortices of schizophrenic patients might be revealed by examining dopamine D1 receptor density in these brain regions. A quantitative autoradiographic assay with [3H]-SCH 23390 was performed with samples from schizophrenic patients, normal controls, neuroleptic-treated controls, and suicides. There was a significant elevation in specific binding of [3H]-SCH 23390 in the intermediate layer of the prefrontal cortex from neuroleptic-treated controls (p = .05). Elevated [3H]-SCH 23390 binding in several layers from prefrontal and cingulate cortex was observed in schizophrenic subjects, although these results did not reach statistical significance. When data from subjects who had received neuroleptics (schizophrenics and neuroleptic controls) were compared to subjects who had not received neuroleptics (normal controls and suicides), there was a significant elevation in receptor density in both the prefrontal (p = .05) and cingulate cortices (p = .03). These data suggest that elevated [3H]-SCH 23390 binding in human prefrontal and cingulate cortices may occur with chronic neuroleptic treatment, although increased receptor density that may exist as a feature of psychotic illnesses cannot be excluded.     

Genetic control of male fertility in Arabidopsis thaliana: structural analyses of postmeiotic developmental mutants

Seven new male-sterile mutants (ms7-ms13) of Arabidopsis thaliana (L.) Heynh. (ecotype columbia) are described that show a postmeiotic defect of microspore development. In ms9 mutants, microspores recently released from the tetrad appear irregular in shape and are often without exines. The earliest evidence of abnormality in ms12 mutants is degeneration of microspores that lack normal exine sculpturing, suggesting that the MS12 product is important in the formation of pollen exine. Teratomes (abnormally enlarged microsporocytes) are also occasionally present and each has a poorly developed exine. In ms7 mutant plants, the tapetal cytoplasm disintegrates at the late vacuolate microspore stage, apparently causing the degeneration of microspores and pollen grains. With ms8 mutants, the exine of the microspores appears similar to that of the wild type. However, intine development appears impaired and pollen grains rupture prior to maturity. In ms11 mutants, the first detectable abnormality appears at the mid to late vacuolate stage. The absence of fluorescence in the microspores and tapetal cells after staining with 4',6-diamidino-2-phenylindole (DAPI) and the occasional presence of teratomes indicate degradation of DNA. Viable pollen from ms10 mutant plants is dehisced from anthers but appears to have surface abnormalities affecting interaction with the stigma. Pollen only germinates in high-humidity conditions or during in-vitro germination experiments. Mutant plants also have bright-green stems, suggesting that ms10 belongs to the eceriferum (cer) class of mutants. However, ms10 and cer6 are non-allelic. The ms13 mutant has a similar phenotype to ms10, suggesting is also an eceriferum mutation. Each of these seven mutants had a greater number of flowers than congenic male-fertile plants. The non-allelic nature of these mutants and their different developmental end-points indicate that seven different genes important for the later stages of pollen development have been identified.     

Structure-activity relationships for 1-phenylbenzimidazoles as selective ATP site inhibitors of the platelet-derived growth factor receptor

1-Phenylbenzimidazoles are shown to be a new class of ATP-site inhibitors of the platelet-derived growth factor receptor (PDGFR). Structure-activity relationships (SARs) are narrow, with closely related heterocycles being inactive. A systematic study of substituted 1-phenylbenzimidazoles showed clear SARs. Substituents at the 4'- and 3'-positions of the phenyl ring are tolerated but do not significantly improve activity, while substituents at the 2'-position abolish it. Substituents in the 2-, 4-, and 7-positions of the benzimidazole ring (with the exception of 4-OH) also abolish activity. Most substituents at the 5- and 6-positions maintain or increase activity, with the 5-OH, 5-OMe, 5-COMe, and 5-CO2Me analogues being >10-fold more potent than the parent 1-phenylbenzimidazole. The 5-OMe analogue was both the most potent inhibitor, and showed the highest selectivity (50-fold) between PDGFR and FGFR isolated enzymes, and also a moderately effective inhibitor (IC50 = 1.9 microM) of PDGF-stimulated PDGFR autophosphorylation in rat aorta smooth muscle cells.