15N-labeled tRNA. Identification of 4-thiouridine in Escherichia coli tRNASer1 and tRNATyr2 by 1H-15N two-dimensional NMR spectroscopy

Uridine is uniquely conserved at position 8 in elongator tRNAs and binds to A14 to form a reversed Hoogsteen base pair which folds the dihydrouridine loop back into the core of the L-shaped molecule. On the basis of 1H NMR studies, Hurd and co-workers (Hurd, R. E., Robillard, G. T., and Reid, B. R. (1977) Biochemistry 16, 2095-2100) concluded that the interaction between positions 8 and 14 is absent in Escherichia coli tRNAs with only 3 base pairs in the dihydrouridine stem. We have taken advantage of the unique 15N chemical shift of N3 in thiouridine to identify 1H and 15N resonances for the imino units of S4U8 and s4U9 in E. coli tRNASer1 and tRNATyr2. Model studies with chloroform-soluble derivatives of uridine and 4-thiouridine show that the chemical shifts of the protons in the imino moieties move downfield from 7.9 to 14.4 ppm and from 9.1 to 15.7 ppm, respectively; whereas, the corresponding 15N chemical shifts move downfield from 157.5 to 162.5 ppm and from 175.5 to 180.1 ppm upon hydrogen bonding to 5'-O-acetyl-2',3'-isopropylidene adenosine. The large difference in 15N chemical shifts for U and s4U allows one to unambiguously identify s4U imino resonances by 15N NMR spectroscopy. E. coli tRNASer1 and tRNATyr2 were selectively enriched with 15N at N3 of all uridines and modified uridines. Two-dimensional 1H-15N chemical shift correlation NMR spectroscopy revealed that both tRNAs have resonances with 1H and 15N chemical shifts characteristic of s4UA pairs. The 1H shift is approximately 1 ppm upfield from the typical s4U8 resonance at 14.8 ppm, presumably as a result of local diamagnetic anisotropies. An additional s4U resonance with 1H and 15N shifts typical of interaction of a bound water or a sugar hydroxyl group with s4U9 was discovered in the spectrum of tRNATyr2. Our NMR results for tRNAs with 3-base pair dihydrouridine stems suggest that these molecules have an U8A14 tertiary interaction similar to that found in tRNAs with 4-base pair dihydrouridine stems.     

Isopentenyl diphosphate:dimethylallyl diphosphate isomerase. An improved purification of the enzyme and isolation of the gene from Saccharomyces cerevisiae

Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase) is an enzyme in the isoprenoid biosynthetic pathway which catalyzes the interconversion of the primary five-carbon homoallylic and allylic diphosphate building blocks. We report a substantially improved procedure for purification of this enzyme from Saccharomyces cerevisiae. An amino-terminal sequence (35 amino acids) was obtained from a highly purified preparation of IPP isomerase. Oligonucleotide probes based on the protein sequence were used to isolate the structural gene encoding IPP isomerase from a yeast lambda library. The cloned gene encodes a 33,350-dalton polypeptide of 288 amino acids. A 3.5-kilobase EcoRI fragment containing the gene was subcloned into the yeast shuttle vector YRp17. Upon transformation with plasmids containing the insert, a 5-6-fold increase in IPP isomerase activity was seen in transformed cells relative to YRp17 controls, confirming the identity of the cloned gene. This is the first reported isolation of the gene for IPP isomerase.     

Cloning an ipt gene from Agrobacterium tumefaciens: characterisation of cytokinins in derivative transgenic plant tissue

The isopentenyl transferase gene was isolated from Agrobacterium tumefaciens AcH5 using polymerase chain reaction and transformed into Petunia and Kalanchoë using both A. tumefaciens and A. rhizogenes transformation systems. Morphological evidence and elevated endogenous cytokinin levels indicated that the PCR product was an active gene. Accurate quantification of the cytokinins was obtained by radioimmunoassay, following purification and separation of the free bases and ribosides by HPLC. Of the six cytokinins quantified, zeatin riboside and its stabilised dihydro-derivative, dihydrozeatin riboside, showed the greatest increases in the transformed Petunia tissue (up to 600-fold). The importance of measuring changes in individual cytokinins is discussed.     

A recombination hot spot in the Rh genes revealed by analysis of unrelated donors with the rare D-- phenotype.

We have studied the arrangement of Rh (rhesus) genes in donors who are completely null for the products of one of them, RHCE. We show that five of six homozygous individuals with the so-called Rh D-- phenotype, who express no red-cell antigens of the C/c and E/e series, have rearranged RHCE genes in which internal sequences have been replaced by the corresponding sequences from RHD. Moreover, although there is heterogeneity at the 3' end, the 5' boundary of this chimerism is within the same small interval around exon 2. This interval is characterized by an exceptionally high degree of sequence homology between RHCE and RHD, a high density of dispersed repetitive elements, and the presence of an alternating purine-pyrimidine copolymer tract. We suggest that these features may explain the mechanistic basis for the origin of the rearrangement.     

Purification and characterization of dimethylallyl tryptophan synthase from Claviceps purpurea.

Dimethylallyl tryptophan synthase (DMAT synthase) catalyzes the alkylation of L-tryptophan by dimethylallyl diphosphate to form 4-(gamma,gamma-dimethylallyl)-L-tryptophan. The enzyme from mycelia of Claviceps purpurea was purified approximately 125-fold to apparent homogeneity by chromatography on n-butyl Sepharose, Q Sepharose, phenyl Sepharose, and Protein Pak as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analysis by gel filtration chromatography and SDS-PAGE indicated that DMAT synthase is an alpha 2 dimer with a molecular mass of 105 kDa. The purified enzyme was active in metal-free buffer containing EDTA. However, activity was enhanced upon addition of divalent calcium or magnesium ions to the buffer. Values for KM and Vmax were determined in the metal-free EDTA buffer (KMDMAPP, 14 microM; KML-tryptophan, 40 microM; Vmax, 215 nmol min-1 mg-1), 4 mM CaCl2 (KMDMAPP, 8.0 microM; KML-tryptophan, 17 microM; Vmax, 504 nmol min-1 mg-1), and 4 mM MgCl2 (KMDMAPP, 8.0 microM; KML-tryptophan, 12 microM; Vmax, 455 nmol min-1 mg-1). The product was isolated and characterized by 1H NMR, uv, and FAB mass spectrometry.     

Changes in lung macrophages during disease

Abstract The alveolar macrophage pool is morphologically and phenotypically heterogenous. Using monoclonal antibodies, homogeneous populations were isolated from broncho-alveolar lavage for functional studies. Such investigations revealed that some alveolar macrophages exhibit phagocytic ad microbicidal capacity, thus being equipped to act as effector cells (MoAb RFD7 +), while othrs exhibit the characteristics of antigen presenting cells (MoAb RFD1 +) involved in the induction of acquired T cell responses. In the investigation of cells from sarcoid patients, a third population was revealed, exhibiting the phenotype RFD1 + and RFD7 +. Functional studies of such cells showed them to act by suppressing T-cell responsiveness. The balance between these three populations alters in different disease processes and, in the case of sarcoidosis, is related to disease severity. It is propose that changes in the balance of these populations may directly influence the pathogenesis of inflammatory lung disease.     

Conservation of genetic linkage in nonisogenic isolates of Candida albicans

A number of laboratories are now engaged in the genetic analysis of Candida albicans. This diploid yeast, the major fungal pathogen of humans, is imperfect. Parasexual techniques have been devised for complementation and recombination analysis in this organism. This paper attempts to address the question of the extent to which nonisogenic strains of C. albicans have conserved a common genetic map. This analysis is a prerequisite for the integration of work done in different laboratories and may also provide useful information on the taxonomy of the genus Candida. The paper also reports the analysis of an interspecific hybrid between C. albicans and Candida stellatoidea. The method employed in these studies was the analysis of the mitotic recombination relationships of a group of linked genes and their centromere. Strains carrying linked auxotrophic mutations were fused with isogenic and nonisogenic complementary strains to form tetraploids. The mitotic recombination analyses of these tetraploids suggest that in the isolates studied the genetic map is conserved. A comparison of tetraploid and diploid mitotic recombination analyses is also presented.     

Isometric force development, isotonic shortening, and elasticity measurements from Ca(2+)-activated ventricular muscle of the guinea pig

Isometric tension and isotonic shortening were measured at constant levels of calcium activation of varying magnitude in mechanically disrupted EGTA-treated ventricular bundles from guinea pigs. The results were as follows: (a) The effect of creatine phosphate (CP) on peak tension and rate of shortening saturated at a CP concentration more than 10 mM; below that level tension was increased and shortening velocity decreased. We interpreted this to mean that CP above 10 mM was sufficient to buffer MgATP(2-) intracellularly. (b) The activated bundles exhibited an exponential stress-strain relationship and the series elastic properties did not vary appreciably with degree of activation or creatine phosphate level. (c) At a muscle length 20 percent beyond just taut, peak tension increased with Ca(2+) concentration over the range slightly below 10(-6) to slightly above 10(-4)M. (d) By releasing the muscle length-active tension curves were constructed. Force declined to 20 percent peak tension with a decrease in muscle length (after the recoil) of only 11 percent at 10(-4)M Ca(2+) and 6 percent at 4x10(-6)M Ca(2+). (e) The rate of shortening after a release was greater at lower loads. At identical loads (relative to maximum force at a given Ca(2+) level), velocity at a given time after the release was less at lower Ca(2+) concentrations; at 10 M(-5), velocity was 72 percent of that at 10(-4)M, and at 4x10(-6)M, active shortening was usually delayed and was 40 percent of the velocity at 10(-4) M. Thus, under the conditions of these experiments, both velocity and peak tension depend on the level of Ca(2+) activation over a similar range of Ca(2+) concentration.     

Dopamine-mediated behaviour and 3H-spiperone binding to striatal membranes in rats after nine months haloperidol administration

Rats were treated with haloperidol (1.5mg/kg/day) in their drinking water for 9 months, with or without a subsequent withdrawal period of 7–10 days. Compared with controls, spontaneous locomotion and apomorphine-induced stereotypy were reduced in rats maintained on haloperidol whereas both behaviours were increased after the withdrawal period. Maximum specific ³H-spiperone binding to striatal membrane preparations was increased (about 65%) in drug-treated rats with or without a withdrawal period. The dissociation constant for ³H-spiperone binding was significantly increased only in those rats maintained on haloperidol with no withdrawal period. The increase in maximum binding of ³H-spiperone was larger than that reported after less prolonged administration of neuroleptics. The size of the change should be taken into account in assessing the increased ligand binding reported in post-mortem brains of schizophrenics.     

Carbon implications of converting cropland to bioenergy crops or forest for climate mitigation: a global assessment

The potential for climate change mitigation by bioenergy crops and terrestrial carbon sinks has been the object of intensive research in the past decade. There has been much debate about whether energy crops used to offset fossil fuel use, or carbon sequestration in forests, would provide the best climate mitigation benefit. Most current food cropland is unlikely to be used for bioenergy, but in many regions of the world, a proportion of cropland is being abandoned, particularly marginal croplands, and some of this land is now being used for bioenergy. In this study, we assess the consequences of land‐use change on cropland. We first identify areas where cropland is so productive that it may never be converted and assess the potential of the remaining cropland to mitigate climate change by identifying which alternative land use provides the best climate benefit: C₄ grass bioenergy crops, coppiced woody energy crops or allowing forest regrowth to create a carbon sink. We do not present this as a scenario of land‐use change – we simply assess the best option in any given global location should a land‐use change occur. To do this, we use global biomass potential studies based on food crop productivity, forest inventory data and dynamic global vegetation models to provide, for the first time, a global comparison of the climate change implications of either deploying bioenergy crops or allowing forest regeneration on current crop land, over a period of 20 years starting in the nominal year of 2000 ad . Globally, the extent of cropland on which conversion to energy crops or forest would result in a net carbon loss, and therefore likely always to remain as cropland, was estimated to be about 420.1 Mha, or 35.6% of the total cropland in Africa, 40.3% in Asia and Russia Federation, 30.8% in Europe‐25, 48.4% in North America, 13.7% in South America and 58.5% in Oceania. Fast growing C₄ grasses such as Miscanthus and switch‐grass cultivars are the bioenergy feedstock with the highest climate mitigation potential. Fast growing C₄ grasses such as Miscanthus and switch‐grass cultivars provide the best climate mitigation option on ≈485 Mha of cropland worldwide with ~42% of this land characterized by a terrain slope equal or above 20%. If that land‐use change did occur, it would displace ≈58.1 Pg fossil fuel C equivalent (C_eq oil). Woody energy crops such as poplar, willow and Eucalyptus species would be the best option on only 2.4% (≈26.3 Mha) of current cropland, and if this land‐use change occurred, it would displace ≈0.9 Pg C_eq oil. Allowing cropland to revert to forest would be the best climate mitigation option on ≈17% of current cropland (≈184.5 Mha), and if this land‐use change occurred, it would sequester ≈5.8 Pg C in biomass in the 20‐year‐old forest and ≈2.7 Pg C in soil. This study is spatially explicit, so also serves to identify the regional differences in the efficacy of different climate mitigation options, informing policymakers developing regionally or nationally appropriate mitigation actions.