Unilateral duplication of the cerebellar hemisphere and internal, middle, and external ear: a clinical case study

This article presents the unique congenital anomaly complex of ipsilateral cerebellar hemisphere duplication and internal, middle, and external ear duplication. Diagnostic techniques included a head CT scan, a three-dimensional head CT scan, and a head MRI. An aberrant notochordal split was proposed as the embryologic mechanism leading to the development of such anomalies.     

Anaerobic degradation of toluene and xylene by aquifer microorganisms under sulfate-reducing conditions.

Toluene and the three isomers of xylene were completely mineralized to CO2 and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was gasoline-contaminated sediment from Seal Beach, Calif. Evidence confirming that sulfate was the terminal electron acceptor is presented. Benzene and ethylbenzene were not degraded under the experimental conditions used. Successive transfers of the mixed cultures that were enriched from aquifer sediments retained the ability to degrade toluene and xylenes. Greater than 90% of 14C-labeled toluene or 14C-labeled o-xylene was mineralized to 14CO2. The doubling time for the culture grown on toluene or m-xylene was about 20 days, and the cell yield was about 0.1 to 0.14 g of cells (dry weight) per g of substrate. The accumulation of sulfide in the cultures as a result of sulfate reduction appeared to inhibit degradation of aromatic hydrocarbons.     

Effects of the steroid antagonist RU486 on dimerization of the human progesterone receptor.

We previously reported, using a coimmunoprecipitation assay, that the B form (PR-B) of the human progesterone receptor from T47D human breast cancer cells dimerizes in solution with the A receptor (PR-A) and that the extent of dimerization correlates with receptor binding activity for specific DNA sequences [DeMarzo, A.M., Beck, C.A., O?ate, S.A., & Edwards, D.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 72-76]. This suggested that solution dimerization is an intermediate step in the receptor activation process. The present study has tested the effects of the progesterone antagonist RU486 on solution dimerization of progesterone receptors (PR). As determined by the coimmunoprecipitation assay, RU486 binding did not impair dimerization of receptors; rather, the antagonist promoted more efficient solution dimerization than the progestin agonist R5020. This enhanced receptor dimerization correlated with a higher DNA binding activity for transformed receptors bound with RU486. RU486 has been shown previously to produce two other alterations in the human PR when compared with R5020. PR-RU486 complexes in solution exhibit a faster sedimentation rate (6 S) on salt-containing sucrose density gradients than PR-R5020 complexes (4 S), and PR-DNA complexes have a faster electrophoretic mobility on gel-shift assays in the presence of RU486. We presently show that the 6 S PR-RU486 complex is a receptor monomer, not a dimer. The increased sedimentation rate and increased mobility on gel-shift assays promoted by RU486 were also observed with recombinant PR-A and PR-B separately expressed in insect cells from baculovirus vectors. These results suggest that RU486 induces a distinct conformational change both in PR monomers in solution and in dimers bound to DNA. We also examined whether conformational changes in PR induced by RU486 would prevent a PR polypeptide bound to RU486 from heterodimerization with another PR polypeptide bound to R5020. To evaluate this, PR-A and PR-B that were separately bound to R5020 or RU486 in whole cells were mixed in vitro. PR-A-RU486 was capable of dimerization with PR-B-R5020, and this was demonstrated for heterodimers both formed in solution and bound to specific DNA. The capability to form heterodimers in vitro raises the possibility that the antagonist action of RU486 in vivo could in part be imposed in a dominant negative fashion through heterodimerization between one receptor subunit bound to an agonist and another bound to RU486.     

Receptor expression and oxidase activity in human neutrophils: Regulation by granulocyte-macrophage colony-stimulating factor and dependence upon protein biosynthesis

Incubation of human bloodstream neutrophils with 50 u/ml recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) primed the respiratory burst (as assessed by fMet-Leu-Phe stimulated luminol-dependent chemiluminescence) and resulted in a rapid (within 15 min) upregulation of expression of CD11b and CD18 (as measured by FACS analysis). This rapid priming and modulation of receptor expression was not inhibited by cycloheximide and hence appeared to be independent ofde novo protein biosynthesis. When neutrophils were incubated for up to 5 h in culture, the fluorescence distributions of CD11b and CD18 declined indicating the loss of expression of these receptors as the neutrophils aged, but in rGM-CSF treated suspensions receptor expression was maintained. When neutrophils were incubated in the presence of cycloheximide, they progressively lost their ability to generate reactive oxidants in response to fMet-Leu-Phe so that by 5 h incubation with this inhibitor they could only generate about 25% of the oxidative response stimulated in untreated cells, and the expression of CD16 and CD18 was grossly impaired. Similar effects were observed in rGM-CSF treated suspensions except that cycloheximide required longer incubation times (typically 4–5 h) before impairment of function or receptor expression occurred. These data show thatde novo protein biosynthesis is required for both the maintenance of neutrophil function and also for the continued expression of some plasma membrane receptors.Abbreviations fMet-Leu-Phe N-formylmethionyl-Leucyl-Phenylalanine - rGM-CSF recombinant granulocyte-macrophage colony-stimulating factor - FITC fluorescein isothiocyanate conjugate - Luminol 5-amino-2,3-dihydrophthalazine-1,4-dione     

Neuropeptide Y inhibits cAMP accumulation in renal proximal convoluted tubules

The effect of neuropeptide Y (NPY) on cAMP accumulation in various segments of the rabbit nephron was examined. NPY inhibited parathyroid hormone-stimulated cAMP accumulation in the proximal convoluted tubule in a concentration-dependent manner. NPY also inhibited forskolin-stimulated cAMP production in this segment of the nephron. In contrast, NPY had no effect on parathyroid hormone or forskolin-stimulated cAMP accumulation in the proximal straight tubule. Similarly, NPY had no effect on forskolin-stimulated cAMP levels along the rest of the nephron. These results are consistent with previous studies which have localized NPY receptors to the proximal convoluted tubule, and suggest that NPY via its effects on cAMP metabolism may play a role in proximal tubule transport.     

Identification of a serum-inducible messenger RNA (5B10) as the mouse homologue of calcyclin: tissue distribution and expression in metastatic, ras-transformed NIH 3T3 cells

A mouse mRNA, provisionally designated 5B10, has been cloned based on its inducibility by serum in quiescent murine fibroblasts. Here we report the full-length complementary DNA sequence and a partial characterization. There are about five copies of the gene in the mouse genome. Sequence analysis of the 5B10 coding region reveals 94 and 97% amino acid identity to human and rat calcyclin, respectively. Although the coding region has been highly conserved during evolution of the rodent and human genomes, the untranslated flanking sequences differ significantly. A protein of Mr about 8000 was produced by in vitro translation of the mRNA transcribed in vitro from 5B10 complementary DNA in a riboprobe vector. An antiserum raised against a portion of the predicted human calcyclin protein cross-reacted with this mouse protein. 5B10 mRNA was found in greatest amount in organs containing proliferating cells, e.g., epidermis, skin, stomach, uterus of pregnant mouse, placenta, and decidua. Brain, liver, mature thymus, and skeletal muscle had little or no detectable 5B10 mRNA. 5B10 mRNA levels were higher in cells treated with 7,12-dimethylbenzanthracene and 12-O-tetradecanoylphorbol-13-acetate than in their normal counterparts, suggesting a role in tumorigenesis. In addition, high 5B10 mRNA levels were associated with metastatic ability in a series of ras-transformed cells, in proportion to levels of ras p21 expressed by the cells, implicating 5B10 even more deeply in carcinogenesis.     

Purification and characterization of an extracellular amylase from a thermophilic streptomycete

G oldberg , J.D. & E dwards , C. 1990. Purification and characterization of an extracellular amylase from a thermophilic streptomycete. Journal of Applied Bacteriology 69 , 712–717.
A single extracellular alpha-amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1) from Streptomyces thermoviolaceus subsp. apingens was purified to homogeneity by a starch adsorption method. SDS-PAGE indicated that the enzyme had an apparent M, of 57 kDa and activity was optimal at a pH of 7–2 and a temperature of 55C. It employed an endo-active mechanism to liberate predominantly maltose, as well as smaller amounts of higher oligosaccharides when incubated with starch. EDTA inhibited enzyme activity, suggesting an involvement of a divalent cation in activity. The enzyme was also stabilized by divalent cations when heated and the results suggested a major role for Ca²⁺ ions for both activity and thermostability. The alpha-amylase from S. thermoviolaceus displayed some similarities with commercially-used streptomycete alpha-amylases.     

Cooperative assembly of EBNA1 on the Epstein-Barr virus latent origin of replication.

The EBNA1 protein of Epstein-Barr virus (EBV) activates DNA replication by binding to multiple copies of its 18-bp recognition sequence present in the Epstein-Barr virus latent origin of DNA replication, oriP. Using electrophoretic mobility shift assays, we have localized the minimal DNA binding domain of EBNA1 to between amino acids 470 and 607. We have also demonstrated that EBNA1 assembles cooperatively on the dyad symmetry subelement of oriP and that this cooperative interaction is mediated by residues within the minimal DNA binding and dimerization domain of EBNA1.     

Translocation of the Retinoblastoma Gene Product during Mitosis

This study reports the immunocytochemical localization of the retinoblastoma gene product within synchronized normal human keratinocytes. Data suggest that mitotic spindles function in the transport of the retinoblastoma tumor suppressor gene product during cell division. A diffuse anti-pRB reactivity was detected within the nuclei of G₁-phase keratinocytes, although staining was not evident within the nucleoli. During S-phase and G₂-phase the anti-pRB reactivity was localized to discrete regions within the nuclear compartment. The anti-pRB reactivity of M-phase cells was localized to the mitotic spindles and microtubule nucleation centers. Immunoprecipitation and Western blotting of the pRB antigen from synchronized keratinocytes showed that the apparent polypeptide molecular weight of pRB increased from 105 kDa during G₁-phase to 115 kDa during M-phase. Immunoprecipitation of the pRB antigen from mitotic spindles resulted in the coprecipitation of two polypeptides with apparent polypeptide molecular weights of 115 and 50 kDa. Western blotting of the immunoprecipitates from purified keratinocyte mitotic spindles showed that β-tubulin was the 50-kDa polypeptide associated with hyperphosphorylated pRB.     

Trinuclear iridium and rhodium complexes: the solution to a puzzle involving the multiple coordination possibilities of 1,8-naphthyridine-2-one ligands

The multiple coordination possibilities of 1,8-naphthyridine-2-one (HOnapy) and 5,7-dimethyl-1,8-napthyridine-2-one (HOMe₂napy) ligands allow the synthesis of a variety of tri- di- and mononuclear complexes, showing fluxional behaviour and frequent exchange of the coordinated ML₂ fragments. Thus, reactions of [M₂(μ-OMe)₂(cod)₂] (cod = 1,5-cyclooctadiene) with HOnapy and HOMe₂napy yield the compounds of the general formula [M(μ-OR₂napy) (cod)]_n (M = Ir, R = Me (1a, 1b, H (2); M = Rh, R = Me (3a, 3b). They crystallise as inconvertible yellow (a) and purple/orange (b) forms and also show a puzzling behaviour in solution. X-ray diffraction studies on both forms (3a, 3b) and spectroscopic data reveal that the yellow forms are mononuclear complexes whilst the dark-coloured crystals contain dinuclear complexes. In solution, the nuclearity of the complexes depends on the solvent. In addition both types of complexes are fluxional. The mixed-ligand complexes [M₂(μ-OMe₂napy)₂(CO)₂(cod)] M = Ir (5), Rh (6) have been isolated and characterised; they are found to be intermediates in the synthesis of the trinuclear complexes [M₃(μ₃-OMe₂napy)₂(CO)₂(cod)₂]⁺ M = Rh (8), Ir (9). Reactions of [IrCl(CO)₂(NH₂-p-tolyl] with the complexes [Rh(μ-OR₂napy)(diolefin)]_n followed by addition of a poor donor anion is a general one-pot synthesis for the hetertrinuclear complexes [Rh₂Ir(μ₃-OR₂napy)₂(CO)₂(diolefin)₂]⁺ (R=Me, DIOLEFIN = cod (10), tetrafluorobenzo-barrelene (tfbb) (11), 2,5-norbornadiene (nbd) (12); R=H, DIOLEFIN=cod (13)). This synthesis follows a stepwise mechanism from the mononuclear to the trinuclear complexes in which mixed-ligand heterodinuclear complexes are involved as intermediates of the type [(diolefin)Rh(μ-OMe₂napy)₂Ir(CO)₂]. Heteronuclear complexes which possess the core [RhIr₂]³⁺, such as [RhIr₂(μ₃-OR₂napy)₂(CO)₂(cod)₂]BF₄ (R=Me (14), H (15)), result from the reaction of 1 or 2 with [Rh(CO)₂S_x]⁺ (S = solvent). The trinuclear complexes undergo two chemically reversible one-electron oxidation processes. The chemical oxidation of 10, 14 and 9 with silver salts gives the mixed-valence trinuclear radicals [Rh₂Ir(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (16), [RhIr₂(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (17) and [Ir₃(μ₃-OMe₂napy)₂(CO)₂(cod)₂]²⁺ (18), which have been isolated as the perchlorate and tetrafluoroborate salts. The EPR spectrum of 16 indicates that the unpaired electron is essentially in an orbital delocalised on the metals. The molecular structures of the complexes 3a, 3b, 6, 10b and 16a are described. Crystals of 3a are triclinic, P-1, with a = 9.7393(2), b = 14.0148(4), c = 16.0607(4) Å, α = 88.122(3), β = 83.924(3), γ = 87.038(3)°, Z = 4; 3b crystallises in the Pna2_i orthorhhombic space group, with a = 16.7541(3), B = 11.7500(8), c = 17.7508(7) Å, Z = 4; complex 6 is packed in the monoclinic space group P2_i/c, a = 9.6371(1), b = 11.8054(4), c = 27.2010(9) Å, β = 90.556(4)°, Z = 4; crystals of 10b are monoclinic, P2₁/n, with a = 17.546(7), b = 13.232(6), c = 17.437(8) Å, β = 106.18(1)°, Z = 4; crystals of 16a are triclinic, P-1, with a = 10.318(4), b = 12.562(6), C = 19.308(8) Å, α = 92.12(8), β = 97.65(9), γ = 90.68(5)°, Z = 2. The five different structures show the coordination versatility of the OMe₂napy molecule as ligand, which behaves as a N,N′-chelating (3a), bidentate N,O-donor (3b, 6), or as a tridentate N,N′,O-donor bridging ligand (10b, 16a).