Construction of a shuttle vector for Zymomonas mobilis

Summary An Escherichia coli-Zymomonas mobilis shuttle vector was constructed from a 15.5 kb native plasmid of ZM6 00 and the E. coli plasmid, pBR329. Integrative transfer of this shuttle vector from E. coli to Z. mobilis was achieved with the aid of the mobilizing plasmid, pRK2013. The shuttle vector was stable in Z. mobilis for at least 300 generations without antibiotic selection.Offprint requests to: S. F. Delaney     

Insulin binding in the hypothalamus of lean and genetically obese Zucker rats

Recent reports have suggested that the obesity and hyperphagia of the genetically obese Zucker rat may be related to defective insulin action or binding in the hypothalamus. We used quantitative autoradiography to determine if insulin binding is altered in specific hypothalamic nuclei associated with food intake. Insulin binding was measured in the arcuate (ARC), dorsomedial (DMN), and ventromedial (VMN) hypothalamic nuclei of 3–4-month-old lean (Fa/Fa) and genetically obese (fa/fa) Zucker rats. A consistently reproducible 15% increase in the total specific binding of 0.1 nM [¹²⁵I]-insulin was found in the ARC of the obese genotype. A slight increase in insulin binding in the DMN was also found. No difference in specific insulin binding was found between genotypes in the VMN. Nonlinear least squares analysis of competitive binding studies showed that the K_d of the ARC insulin binding site was 33% higher in the lean rats than in the obese rats, indicating an increased affinity for insulin. No difference in site number (B_max) was found in the ARC, DMN or VMN, and no evidence was found for reduced insulin binding in the hypothalamus of the obese (fa/fa) genotype. The results suggest that hyperphagia and obesity of the obese (fa/fa) Zucker rat genotype may be associated with increased insulin binding in the arcuate nucleus.     

Murine embryonal carcinoma cell-surface sialyl Le is present on a novel glycoprotein and on high-molecular-weight lactosaminoglycan

Carbohydrate-specific monoclonal antibodies were used to demonstrate the expression of a new membrane glycoprotein on F9 murine embryonal carcinoma cells. Sialyl Le^x was detected using monoclonal antibody FH6 in a sensitive, cell monolayer radioimmunoassay. The antigen codistributed in gel filtration of a crude homogenate and in a membrane-enriched fraction with two known lactosaminoglycan markers, i and SSEA-1 (Le^x or X hapten). Sialyl Le^x was further shown to be carried by a novel glycoprotein, termed small lactosaminoglycan-like glycoprotein (sLAG) which could be purified by immunoaffinity chromatography. In two-dimensional polyacrylamide gel electrophoresis this glycoprotein had an apparent molecular weight of 45 kDa and a pI of about 6.5. The more differentiated cell line PYS-2 also expressed sialyl Le^x and i antigens but not Le^x, and FH6-reactive sLAG could be extracted from PYS-2 membranes. Sialylation of fucosylated type 2 carbohydrate chains (X haptens) thus may be an early modification of embryonic carbohydrate antigens.     

Novel Pathway of Toluene Catabolism in the Trichloroethylene-Degrading Bacterium G4

o-Cresol and 3-methylcatechol were identified as successive transitory intermediates of toluene catabolism by the trichloroethylene-degrading bacterium G4. The absence of a toluene dihydrodiol intermediate or toluene dioxygenase and toluene dihydrodiol dehydrogenase activities suggested that G4 catabolizes toluene by a unique pathway. Formation of a hybrid species of ¹⁸O- and ¹⁶O-labeled 3-methylcatechol from toluene in an atmosphere of ¹⁸O₂ and ¹⁶O₂ established that G4 catabolizes toluene by successive monooxygenations at the ortho and meta positions. Detection of trace amounts of 4-methylcatechol from toluene catabolism suggested that the initial hydroxylation of toluene was not exclusively at the ortho position. Further catabolism of 3-methylcatechol was found to proceed via catechol-2,3-dioxygenase and hydroxymuconic semialdehyde hydrolase activities.     

Bacteriophage Transport in Sandy Soil and Fractured Tuff

Bacteriophage transport was investigated in laboratory column experiments using sandy soil, a controlled field study in a sandy wash, and laboratory experiments using fractured rock. In the soil columns, the phage MS-2 exhibited significant dispersion and was excluded from 35 to 40% of the void volume but did not adsorb. Dispersion in the field was similiar to that observed in the laboratory. The phage f2 was largely excluded from the porous matrix of the two fractured-rock cores studied, coming through 1.2 and 2.0 times later than predicted on the basis of fracture flow alone. Because of matrix diffusion, nonsorbing solutes were retarded by over a factor of three relative to fracture flow. The time for a solute tracer to equilibrate with the porous matrix of 6.5-cm-diameter by 25-cm-long cores was measured in days. Results of both granular-medium and fractured-rock experiments illustrate the inability of a solute tracer to provide estimates for dispersion and effective porosity that are applicable to a colloid. Bacteriophage can be used to better estimate the maximum subsurface transport rate of colloidal contaminants through a porous formation.     

Fe2+ binding to apo and holo mammalian ferritin

The binding of Fe2+ to both apo and holo mammalian ferritin has been investigated under anaerobic conditions as a function of pH. In the pH range 6.0-7.5, 8.0 +/- 0.5 Fe2+ ions bind to each apoferritin molecule, but above pH 7.5, a pH-dependent Fe2+ binding profile is observed with up to 80 Fe2+ ions binding at pH 10.0. This Fe2+ binding is reversible and is accompanied by up to two H+ being released per Fe2+ bound at pH 10.0. The Fe2+ binding to apoferritin probably occurs in the 3-fold channels. A much larger and more complex pH-dependent Fe2+ binding stoichiometry was observed for holoferritin with up to 300 Fe2+ ions binding at pH 10.0. This pH-dependent Fe2+ binding was interpreted as Fe2+ interaction at the FeOOH mineral surface with displacement of H+ from -OH or phosphate surface groups by the incoming Fe2+ ions. Mossbauer spectroscopic measurements using 57Fe-labeled Fe2+ under anaerobic conditions showed that 57Fe2+ binding to holoferritin was accompanied by electron transfer to the core, yielding 57Fe3+, presumably bound to the mineral surface. Removal of added iron by Fe2+-specific chelating agents yielded 57Fe2+, demonstrating the reversibility of this electron-transfer process. The Fe2+ bound to apo- and holoferritin is readily converted to Fe3+ by exposure to O2 and strongly retained by the respective ferritin species.     

Redox reactions associated with iron release from mammalian ferritin

The early redox events involved in iron reduction and mobilization in mammalian ferritin have been investigated by several techniques. Sedimentation velocity measurements of ferritin samples with altered core sizes, prepared by partial reduction and Fe2+ chelation, suggest two different events occur during iron loss from the ferritin core. Reductive optical titrations confirm this biphasic behavior by showing that the first 20-30% of core reduction has different optical properties than the latter 70-80%. Proton uptake during initial core reduction is near zero, but as the percent core reduction increases, the proton uptake (H+/e) values increase to 2 H+/e (2 H+/Fe3+ reduced) as core reduction approaches 1 e/Fe3+. Coulometric reduction of ferritin by mediators of different redox potential and different cross-sectional areas show a two-phase sigmoidal reaction pattern in which initial core reduction occurs at a slower rate than later core reduction. The above experiments were all conducted in the absence of iron chelators so that the observed results were all attributed to core reduction rather than the combined effects of core reduction accompanied by Fe2+ chelation. The coulometric reduction of ferritin by various mediators shows a correlation more with reduction potential than with molecular cross-sectional area. The role of the ferritin channels in core reduction is considered in terms of the reported results.     

Proton nuclear magnetic resonance characterization of the aromatic residues in the variant-3 neurotoxin from Centruroides sculpturatus Ewing

The amino acid sequence for the variant-3 (CsE-v3) toxin from the venom of the scorpion Centruroides sculpturatus Ewing contains eight aromatic residues. By use of 2D NMR spectroscopic methods, the resonances from the individual protons (NH, C alpha H, C beta H',H", and the ring) for each of the individual aromatic residues have been completely assigned. The spatial arrangement of the aromatic ring systems with respect to each other has been qualitatively analyzed by 2D-NOESY techniques. The results show that Trp-47, Tyr-4, and Tyr-42 are in close spatial proximity to each other. The NOESY contacts and the ring current induced shifts in the resonances of the individual protons of Tyr-4 and Trp-47 suggest that the aromatic ring planes of these residues are in an orthogonal arrangement. In addition, the spatial proximity of the rings in the pairs Tyr-4, Tyr-58; Tyr-42, Tyr-40; and Tyr-40, Tyr-38 has also been established. A comparison with the published crystal structure suggests that there is a minor rearrangement of the aromatic rings in the solution phase. No 2D-NOESY contacts involving Phe-44 and Tyr-14 to any other aromatic ring protons have been observed. The pH dependence of the aromatic ring proton chemical shifts has also been studied. These results suggest that the Tyr-58 phenolic group is experiencing a hydrogen-bonding interaction with a positively charged group, while Tyr-4, -14, -38, and -40 are experiencing through-space interactions with proximal negatively charged groups. The Trp-47 indole NH is interacting with the carboxylate groups of two proximal acidic residues. These studies define the microenvironment of the aromatic residues in the variant-3 neurotoxin in aqueous solution.     

Genetic evidence for α2-adrenergic receptor subtypes in rat brain,heart and adrenal gland

Summary A complementary DNA (cDNA) clone - cA₂-47 - corresponding to a new ₂-adrenergic receptor subtype has been isolated from a rat brain cDNA library and used as a hybridization probe to scrutinize the ₂-receptor poly(A⁺) RNAs in rat brain, heart and adrenal gland. Hybridization of the 5 half of the coding region of this cDNA at 37°C to rat brain poly(A+) RNA revealed a single band at 5.8 kb as the size of its corresponding mRNA. Under identical hybridization conditions, a human platelet ₂-receptor genomic probe failed to hybridize to any rat brain mRNAs.Under lower stringency conditions, hybridization of the full-length cDNA, cA₂-47, to selected rat tissue poly(A⁺) RNA showed the presence of four different sized mRNAs in brain and three in both heart and adrenal gland. Messages of 1.3 kb and 2.1 kb were common in all three tissues (although the band at 2.1 kb was slightly higher in the heart and adrenal gland). A 5.8 kb mRNA was unique to the brain and a slightly higher band at 6.0 kb was consistently present in heart and adrenal gland but was absent in the brain. A fourth message at 3.4 kb was found predominantly in the brain and was either absent or present at very low levels in the other tissues examined. Under the same conditions, a human platelet ₂-receptor probe hybridized to similar sized messages of 2.1 and 5.8 kb in rat brain and 2.2 and 6.0 kb in rat heart and adrenal gland. This probe, however, failed to detect the abundant 1.3 kb mRNA common to all tissues or the 3.4 kb message in rat brain. The extent of homology of these messages with cA₂-47 is not confined to limited regions of the cDNA since similar hybridization patterns were observed using either 5-noncoding or 5-coding regions of the probe.These results provide the first direct evidence of a surprisingly large range of mRNA sizes for members of the ₂-receptor family in brain, heart, and adrenal gland. The unique nature of certain members of the family in each of the tissues examined raises the curious possibility that these members might contribute to some of the individualized functions of the brain, cardiovasculature and adrenal gland.     

Growth characteristics and ion contents of non-selected and salt-selected callus lines of highbush blueberry (Vacdnium corymbosum) cultivars Blue Crop and Denise Blue

Non-selected and sodium chloride selected callus lines of Vacdnium corymbosum L.cv Blue Crop and cv. Denise Blue were grown on media supplemented with 0–100 mM NaCl. For both cultivars, fresh weight and dry weight yields were greater in selected lines on all levels of NaCl. Selected lines of Blue Crop displayed better growth than selected lines of Denise Blue at most concentrations of NaCl. Internal Na⁺ and Cl^– concentrations in selected and non-selected lines of both cultivars increased as external concentration was raised. However, selected lines of Blue Crop and Denise Blue accumulated more Na⁺ and Cl^– than non-selected lines. Selected lines of both cultivars maintained higher levels of K⁺ than non-selected lines on all external NaCl levels. Selected lines of Blue Crop had higher levels of Na⁺ and Cl^– than that of Denise Blue. The results suggest Na⁺ and Cl^– accumulation could be a mechanism allowing better growth in selected lines at moderate salinity levels (50–75 mM NaCl).