Application of high-performance liquid chromatography to competitive labeling studies: The chemical properties of functional groups of glucaton

A competitive-labeling study of glucagon was carried out using [³H]- and [¹⁴C]-1-fluoro-2,4-dinitrobenzene to determine simultaneously the chemical properties of the α-amino and imidazole groups of the N-terminal histidine residue, and the lysine and tyrosine residues, under conditions where glucagon is in its physiologically active monomer form. The dinitrophenyl derivatives of these groups were purified by high-performance liquid chromatography which greatly simplified the separation steps of the procedure. The results showed the α-amino and tyrosine groups to have relatively normal behavior, with pK values of 7.98 and 10.22, respectively, while the lysine had a low pK of 8.46. The imidazole function had an apparent pK of 7.84, substantially higher than previous estimates. This difference may be accounted for by the effect of the charged form of the adjacent α-amino group on the nucleophilicity of the imidazole group.     

Ethylene receptor expression is regulated during fruit ripening, flower senescence and abscission

Using theArabidopsis ethylene receptorETR1 as a probe, we have isolated a tomato homologue (tETR) from a ripening cDNA library. The predicted amino acid sequence is 70% identical toETR1 and homologous to a variety of bacterial two component response regulators over the histidine kinase domain. Sequencing of four separate cDNAs indicates that tETR lacks the carboxyl terminal response domain and is identical to that encoded by the tomatoNever ripe gene. Ribonuclease protection showed tETR mRNA was undetectable in unripe fruit or pre-senescent flowers, increased in abundance during the early stages of ripening, flower senescence, and in abscission zones, and was greatly reduced in fruit of ripening mutants deficient in ethylene synthesis or response. These results suggest that changes in ethylene sensitivity are mediated by modulation of receptor levels during development.     

Brain S-Adenosylmethionine Levels Are Severely Decreased in Alzheimer's Disease

Abstract: S -Adenosylmethionine is an essential ubiquitous metabolite central to many biochemical pathways, including transmethylation and polyamine biosynthesis. Reduced CSF S -adenosylmethionine levels in Alzheimer's disease have been reported; however, no information is available regarding the status of S -adenosylmethionine or S -adenosylmethionine-dependent methylation in the brain of patients with this disorder. S -Adenosylmethionine concentrations were measured in postmortem brain of 11 patients with Alzheimer's disease. We found decreased levels of S -adenosylmethionine (−67 to −85%) and its demethylated product S -adenosylhomocysteine (−56 to −79%) in all brain areas examined (cerebral cortical subdivisions, hippocampus, and putamen) as compared with matched controls (n = 14). S -Adenosylmethionine and S -adenosylhomocysteine levels were normal in occipital cortex of patients with idiopathic Parkinson's disease (n = 10), suggesting that the decreased S -adenosylmethionine levels in Alzheimer's disease are not simply a consequence of a chronic, neurodegenerative condition. Reduced S -adenosylmethionine levels could be due to excessive utilization in polyamine biosynthesis. The severe reduction in levels of this essential biochemical substrate would be expected to compromise seriously metabolism and brain function in patients with Alzheimer's disease and may provide the basis for the observations of improved cognition in some Alzheimer's patients following S -adenosylmethionine therapy.     

Tracing lake trophic history with a chironomid–total phosphorus inference model

1. In the absence of historical water chemistry data, predictive biological indicator groups preserved in lake sediments can be employed to reconstruct the history of lake eutrophication. Diatoms are well established in this role, but to augment diatom‐based inferences of nutrient status we investigate the potential use of chironomid midges (Insecta: Chironomidae). 2. Canonical correspondence analysis (CCA) of modern chironomid assemblages in surface sediments from 44 lakes in the English Midlands and Wales, U.K., shows that five environmental variables (total phosphorus (TP), bottom dissolved oxygen, maximum lake depth, Secchi depth and surface water temperature) make a statistically significant (P < 0.05) contribution to explaining the variance in the chironomid data, of which TP makes the largest contribution (29%). 3. The relationship is used to develop a series of weighted averaging (WA) and partial least squares (PLS), (WA‐PLS) models to infer log₁₀TP. The models are evaluated by leave‐one‐out (jack‐knifing) cross‐validation. The simplest minimal adequate model is provided by WA with unweighted inverse deshrinking of root mean square error of prediction (RMSEP_jack=0.34 and r²_jack=0.60). 4. Using this model, the trophic history of Betton Pool, Shropshire, U.K., is reconstructed from the mid‐19th century to the present day and the results from the chironomid‐TP model are compared with inferences from a diatom‐TP model ( 13 ). Both reconstructions suggest that there was a gradual rise in TP since 1850 AD until about 1974, followed by a more pronounced and rapid increase that has continued until the present. Inferred TP values from the WA chironomid inference model agree with diatom‐inferred values. 5. The study demonstrates that fossil chironomid assemblages can be used to investigate quantitatively the trophic history of lakes.     

Photosynthetic and Phylogenetic Primers for Detection of Anoxygenic Phototrophs in Natural Environments

Primer sets were designed to target specific 16S ribosomal DNA (rDNA) sequences of photosynthetic bacteria, including the green sulfur bacteria, the green nonsulfur bacteria, and the members of the Heliobacteriaceae (a gram-positive phylum). Due to the phylogenetic diversity of purple sulfur and purple nonsulfur phototrophs, the 16S rDNA gene was not an appropriate target for phylogenetic rDNA primers. Thus, a primer set was designed that targets the pufM gene, encoding the M subunit of the photosynthetic reaction center, which is universally distributed among purple phototrophic bacteria. The pufM primer set amplified DNAs not only from purple sulfur and purple nonsulfur phototrophs but also from Chloroflexus species, which also produce a reaction center like that of the purple bacteria. Although the purple bacterial reaction center structurally resembles green plant photosystem II, the pufM primers did not amplify cyanobacterial DNA, further indicating their specificity for purple anoxyphototrophs. This combination of phylogenetic- and photosynthesis-specific primers covers all groups of known anoxygenic phototrophs and as such shows promise as a molecular tool for the rapid assessment of natural samples in ecological studies of these organisms.     

Extrachromosomal Recombinant Adeno-Associated Virus Vector Genomes Are Primarily Responsible for Stable Liver Transduction In Vivo

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Although the vector genomes are found both as extrachromosomes and as chromosomally integrated forms in hepatocytes, the relative proportion of each has not yet been clearly established. Using an in vivo assay based on the induction of hepatocellular regeneration via a surgical two-thirds partial hepatectomy, we have determined the proportion of integrated and extrachromosomal rAAV genomes in mouse livers and their relative contribution to stable gene expression in vivo. Plasma human coagulation factor IX (hF.IX) levels in mice originating from a chromosomally integrated hF.IX-expressing transposon vector remained unchanged with hepatectomy. This was in sharp contrast to what was observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein injection of a series of hF.IX-expressing rAAV vectors. At doses of 2.4 x 10(11) to 3.0 x 10(11) vector genomes per mouse (n = 12), hF.IX levels and the average number of stably transduced vector genomes per cell decreased by 92 and 86%, respectively, after hepatectomy. In a separate study, one of three mice injected with a higher dose of rAAV had a higher proportion (67%) of integrated genomes, the significance of which is not known. Nevertheless, in general, these results indicate that, in most cases, no more than approximately 10% of stably transduced genomes integrated into host chromosomes in vivo. Additionally, the results demonstrate that extrachromosomal, not integrated, genomes are the major form of rAAV in the liver and are the primary source of rAAV-mediated gene expression. This small fraction of integrated genomes greatly decreases the potential risk of vector-related insertional mutagenesis associated with all integrating vectors but also raises uncertainties as to whether rAAV-mediated hepatic gene expression can persist lifelong after a single vector administration.     

Sample preconditioning for measurement of fluorescence induction of chlorophyll a in marine phytoplankton

Conditions for measuring fluorescence induction curves (time-scalems) of in vivo chlorophyll ^a were studied using cultures ofDunaliella tertiolecta Butcher (Chlorophyceae) and of Thalassiosirapseudonana Hustedt (3H) (Bacillariophyceae), and samples ofnatural phytoplankton populations from the Grand Banks. Thearea above the fluorescence induction curve (A_DCMU) and themaximum fluorescence intensity (F_max) measured in the presenceof 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) were computedby microcomputer. Cells must be ‘conditioned’ or‘adapted’ prior to obtaining a fluorescence inductioncurve; dark-adaptation resulted in a lower A_DCMU and F_max thandid adaptation in far-red (720 nm) light, and was the conditioningmethod chosen. A_DCMU and F_max increased linearly with increasingirradiance up to 32.8 W m^–2 the highest actinic irradianceavailable. Information on the light history of D. tertiolectawas obtained by following the time-course of change in A_DCMUand in F_max for cells exposed for 10 min to far-red or to bluelight. The rise-time of the fluorescence induction curve andvalues of F_max were greater for samples of D. tertiolecta concentratedonto glass-fiber filters than for liquid samples, however, valuesof A_DCMU for filtered and liquid samples were not significantlydifferent. Samples of Grand Banks phytoplankton collected ontoglass-fiber filters and frozen for 28 d exhibited a significantdecrease in F_max and in A_DCMU relative to the same freshly-filteredsamples. Filtration and freezing of samples is not recommended. ^*This paper is the result of a study made at the Group for AquaticPrimary Productivity (GAP). Second International Workshop heldat the National Oceanographic Institute. Haifa. Israel in April–May1984.     

THE CONSEQUENCES OF BEING DIFFERENT: SINISTRAL COILING IN CERION

The overwhelming predominance of dextral coiling in gastropods is an outstanding and puzzling phenomenon. A few sinistral specimens (left coiling individuals) have been found in many dextral species. Only six sinistral shells have ever been found in Cerion; we base this analysis on the five available shells. We ask whether reversed symmetry is a simple either-or switch without further consequences for shell form, or whether sinistrality engenders associated effects, making left-coiling shells unlike their dextral deme-mates in other ways. All five sinistral shells differ in features of size and coiling late in growth, leading to relatively small apertures and a slight twist in the axis of coiling. We detect and measure this effect as follows: in multivariate morphospace, sinistrals occupy peripheral positions among their dextral deme-mates; in univariate analysis, sinistrals are consistently different for a set of characters involving covariance patterns never before seen in a decade of studies on ontogenetic and age-standardized variation in dextrals; a bootstrap procedure does not recover similar patterns in randomly constituted samples of dextrals matching the true sinistral distribution; direct x-ray measures of the coiling axis detect its slight twist in sinistrals. We discuss the implications of these unsuspected associations for the issues of developmental constraint upon the evolution of morphology.     

An interspecies comparison of normal levels of glycosylated hemoglobin and glycosylated albumin

Aminophenylboronic acid affinity chromatography was used to measure glycosylated hemoglobin and glycosylated albumin levels in a variety of species. The highest glycosylated hemoglobin levels were found in man, the lowest in the chicken and the pig. The highest glycosylated albumin levels were found in avian species, the lowest in the mouse and the rat. A simple kinetic model was used to analyze the rates of formation of glycosylated hemoglobin and albumin in the various species. Rates of glycosylated albumin formation were very similar across the species while rates of glycosylated hemoglobin formation were quite different, presumably reflecting wide differences in erythrocyte permeability to glucose among the species.