Phosphorylation of the GABAA receptor by cAMP-dependent protein kinase and by protein kinase C: Analysis of the substrate domain

Previous work has shown that the GABA_A-receptor (GABA_A-R) could be phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a receptor associated kinase. However, no clear picture has yet emerged concerning the particular subunit subtypes of the GABA_A-R that were phosphorylated by PKA and PKC. In the present report we show that an antibody raised against a 23 amino acid polypeptide corresponding to a sequence in the putative intracellular loop of the 1 subunit of the receptor blocks the in vitro phosphorylation of the purified receptor by PKA and PKC. Moreover, N-terminal sequence analysis of the principal phosphopeptide fragment obtained after proteolysis of the receptor yielded a sequence that corresponds to the 3 subunit of the receptor. Such data provide additional support for our hypothesis (Browning et al., 1990, Proc. Natl. Acad. Sci. USA 87:1315–1317) that both PKA and PKC phosphorylate the -subunit of the GABA_A-R.Special issue dedicated to Dr. Paul Greengard.     

TESTING INFERENCES ABOUT MICRO-EVOLUTIONARY PROCESSES BY MEANS OF SPATIAL AUTOCORRELATION ANALYSIS

We generated numerous simulated gene-frequency surfaces subjected to 200 generations of isolation by distance with, in some cases, added migration or selection. From these surfaces we assembled six data sets comprising from 12 to 15 independent allele-frequency surfaces, to simulate biologically plausible population samples. The purpose of the study was to investigate whether spatial autocorrelation analysis will correctly infer the microevolutionary processes involved in each data set. The correspondence between the simulated processes and the inferences made concerning them is close for five of the six data sets. Errors in inference occurred when the effect of migration was weak, due to low gene frequency differential or low migration strength; when selection was weak and against a background with a complex pattern; and when a random process—isolation by distance—was the only one acting. Spatial correlograms proved more sensitive to detecting trends than inspection of gene-frequency surfaces by the human eye. Joint interpretation of the correlograms and their clusters proved most reliable in leading to the correct inference. The inspection and clustering of surfaces were useful for determining directional components. Because this method relies on common patterns across loci, as many gene frequencies as feasible should be used. We recommend spatial autocorrelation analysis for the detection of microevolutionary processes in natural populations.     

Phylogeny and adaptive radiation within the Anomopoda: a preliminary exploration

The distinctness of the Anomopoda and the polyphyletic nature of the so-called Cladocera are emphasized.An attempt is made to reconstruct the ancestral anomopod, which probably lived in Palaeozoic times. This task is facilitated by the availability of detailed information on extant forms, which includes functional as well as purely morphological considerations and enables us to understand the means whereby complex mechanisms were transformed during evolution. Comparative studies on the ecology and habits of extant forms also throw light on the probable way of life of the ancestral anomopod.Adaptive radiation within the Anomopoda is briefly surveyed and an outline of the suggested phylogeny of the order is indicated.Institute of Environmental and Biological Sciences, University of Lancaster     

Molecular evolution of plant β-glucan endohydrolases

The evolutionary relationships of two classes of plant β-glucan endohydrolases have been examined by comparison of their substrate specificities, their three-dimensional conformations and the structural features of their corresponding genes. These comparative studies provide compelling evidence that the (1→3)-β-glucanases and (1→3,1→4)-β-glucanases from higher plants share a common ancestry and, in all likelihood, that the (1→3,1→4)-β-glucanases diverged from the (1→3)-β-glucanases during the appearance of the graminaceous monocotyledons. The evolution of (1→3,1→4)-β-glucanases from (1→3)-β-glucanases does not appear to have invoked ‘modular’ mechanisms of change, such as those caused by exon shuffling or recombination. Instead, the shift in specificity has been acquired through a limited number of point mutations that have resulted in amino acid substitutions along the substrate-binding cleft. This is consistent with current theories that the evolution of new enzymic activity is often achieved through duplication of the gene encoding an existing enzyme which is capable of performing the required chemistry, in this case the hydrolysis of a glycosidic linkage, followed by the mutational alteration and fine-tuning of substrate specificity. The evolution of a new specificity has enabled a dramatic shift in the functional capabilities of the enzymes. (1→3)-β-Glucanases that play a major role, inter alia, in the protection of the plant against pathogenic microorganisms through their ability to hydrolyse the (1→3)-β-glucans of fungal cell walls, appear to have been recruited to generate (1→3,1→4)-β-glucanases, which quite specifically hydrolyse plant cell wall (1→3,1→4)-β-glucans in the graminaecous monocotyledons during normal wall metabolism. Thus, one class of β-glucan endohydrolase can degrade β-glucans in fungal walls, while the other hydrolyses structurally distinct β-glucans of plant cell walls. Detailed information on the three-dimensional structures of the enzymes and the identification of catalytic amino acids now present opportunities to explore the precise molecular and atomic details of substrate-binding, catalytic mechanisms and the sequence of molecular events that resulted in the evolution of the substrate specificities of the two classes of enzyme.     

Studies in cryoprotection II: Propylene glycol and glycerol

Propylene glycol (PG), glycerol (G), or a combination of both were introduced into and eluted from 116 rabbit kidneys. The function of the kidneys was then studied on an ex vivo shunt by determining bloodflow and creatinine clearance. The cryoprotective agents (CPAs) were introduced and eluted at the rate of about 30 mM/min in an albumin-Ringer's lactate perfusate. In this system, 1.75 to 2.25 M cryoprotectant could be introduced and removed with no demonstrable loss of function. Significant injury was incurred at and above 2.5 M concentrations. When a combination of PG and G (equal proportions) was utilized, both 2.5 and 2.75 M total concentrations were tolerated by the kidney, with no loss of function. These studies demonstrate that in this very rigorous test system, cryoprotectant-induced injury can be significantly diminished by combining two agents of low toxicity. This suggests that osmotic injury may be enhanced by specific toxicities of cryoprotective agents, which can be minimized by using combinations, such as was done in these studies. Also, it has been shown that PG is at least as low in toxicity in the rabbit kidney as is G.     

Chromosomal analyses in vinyl chloride-exposed workers

Chromosomal morphology from cultured peripheral lymphocytes was studied in 81 men; 57 of the men were employed on plants manufacturing vinyl chloride or polyvinylchloride, 19 were on-site controls and 5 were off-site controls. There was a significant increase in chromosomal abnormalities in the exposed workers when compared with the controls. The greatest statistically significant increase in total B and total C cells occurred in autoclave operators, with smaller increases in other job categories. The increase in chromosomal aberrations was correlated with the length of exposure and with a history during the year prior to sampling (1973–1974) of exposure to excursion levels of vinyl chloride. Information on smoking habits was obtained 18 months after blood sampling and a positive correlation between these and total C cell abnormalities was found. There was no positive correlation with various other parameters (bilirubin, platelets, γ-glutamyltranspeptidase, alkaline phosphatase, alanine transaminase and aspartate transaminase). It was not possible to estimate which of the three parameters (smoking history, length of employment or exposure to excursion levels) was the most important.     

A rapid whole-cell assay for superoxide dismutase

A procedure is described for the intact-cell assay of superoxide dismutase(s). The technique involves the use of toluene which renders the cells permeable to the necessary components of a photochemical assay for superoxide dismutase. Whole-cell superoxide dismutase activities from a number of procaryotic and eucaryotic microorganisms compare with cell-free activities and with activities reported in the literature. Using this procedure, changing levels of superoxide dismutase are readily monitored under conditions known to modulate superoxide dismutase activity assayed in vitro. In whole cells of Escherichia coli, exogenous methyl viologen causes a marked increase in superoxide dismutase activity, whereas in the cyanobacterium, Microcystis aeruginosa, such treatment leads to a marked, light-dependent loss of whole-cell superoxide dismutase activity.     

A Note on the Purification of Alcian Blue

Alcian blue 8GX is a copper phthalocyanin dye that shows a high degree of specificity for polyanionic substances such as hyaluronic acid, sialic acid and the chondroitin sulfates. This dye has proved useful for both histochemical and electrophoretic staining of these substances. The Biological Stain Commission has recently begun to certify Alcian blue (Schenk 1981). Commercially available lots contain approximately 50% dye. The remaining constituents have been identified as primarily boric acid, as well as sulfates and dextrins (Scott 1972, Horobin and Goldstein 1972). Horobin and Goldstein (1972) have pointed out that these contaminants may adversely affect staining in the critical electrolyte concentration procedure. Scott (1972), while not ascribing any adverse effects to the presence of boric acid, recommends its removal by differential precipitation with acetone. In this procedure one part of a 2-5% aqueous solution of the dye is added to 5-10 parts of acetone. The precipitated dye is approximately 80% pure. While this method is relatively simple, it does have several drawbacks. Low concentrations of Alcian blue (i.e., 2%) must be used to obtain purities near 80%. Thus a minimum of 250 ml of acetone is needed to purify 1 gram of dye. Furthermore, Horobin and Goldstein (1972) have reported that contamination by dextrin or unknown organic substances (detergent?) interferes with precipitation of the dye enough to make purification by Scott's method impossible. When difficulty in the precipitation of Alcian blue by Scott's method was encountered, the following simple method for the purification of the dye was developed.