Purification and characterisation of an intracellular carbonic anhydrase from the unicellular green alga Coccomyxa

An intracellular carbonic anhydrase (CA; EC 4.2.1.1) was purified and characterised from the unicellular green alga Coccomyxa sp. Initial studies showed that cultured Coccomyxa cells contain an intracellular CA activity around 100 times higher than that measured in high-CO₂-grown cells of Chlamydomonas reinhardtii CW 92. Purification of a protein extract containing the CA activity was carried out using ammonium-sulphate precipitation followed by anion-exchange chromatography. Proteins were then separated by native (non-dissociating) polyacrylamide gel electrophoresis, with each individual protein band excised and assayed for CA activity. Measurements revealed CA activity associated with two discrete protein bands with similar molecular masses of 80 +5 kDa. Dissociation by denaturing polyacrylamide gel electrophoresis showed that both proteins contained a single polypeptide of 26 kDa, suggesting that each 80-kDa native protein was a homogeneous trimer. Isoelectric focusing of the 80-kDa proteins also produced a single protein band at a pH of 6.5. Inhibition studies on the purified CA extract showed that 50% inhibition of CA activity was obtained using 1 M azetazolamide. Polyclonal antibodies against the 26-kDa CA were produced and shown to have a high specific binding to a single polypeptide in soluble protein extracts from Coccomyxa cells. The same antiserum, however, failed to cross-react with soluble proteins isolated from two different species of green algae, Chlamydomonas reinhardtii and Chlorella vulgaris. Correspondingly, antisera directed against pea chloroplastic CA, extracellular CA from C. reinhardtii and human CAII, showed no cross-hybridisation to the 26-kDa polypeptide in Coccomyxa. The 26-kDa protein was confirmed as being a CA by N-terminal sequencing of two internal polypeptide fragments and alignment of these sequences with that of previously identified CA proteins from several different species.Abbreviations CA carbonic anhydrase - CCM CO₂-concentrating mechanism - IEF isoelectric focusing - Rubisco ribulose-l,5-bisphosphate carboxylase/oxygenase We would like to thank Drs. Cecilia Forsman, Inga-Maj Johansson and Nalle Jonsson for their valuable advice concerning the isolation of CA. This work was supported by the Swedish Natural Research Council and Seth M. Kempes Memorial foundation.     

The yellow-cheeked gibbon (Hylobates gabriellae) in nam bai cat tien (southern vietnam) revisited

Data concerning the status, habitat, and vocalizations of yellow-cheeked crested gibbons (Hylobates gabriellae) were collected during a short field trip to the Nam Bai Cat Tien National Park (southern Vietnam). Nam Bai Cat Tien may be the southernmost locality where crested gibbons (i.e. theHylobates concolor group) still survive. Fewer songs were heard at Nam Bai Cat Tien National Park than at other crested gibbon sites visited by the author. At least two gibbon groups appear to have been greatly reduced in number since previous surveys in the park. There is some evidence that both the gibbon population and the gibbon habitat in Nam Bai Cat Tien are disturbed. The first case of a great call solo song in wild gibbons of theconcolor group is reported. Great calls ofH. gabriellae are described and documented with sonagrams for the first time. They differ from those previously described forH. leucogenys.     

Organization of the mitochondrial Cob 2 pseudogene in different lines of rice

In the fertile rice line IR 36 there are two copies of the apocytochrome b (cob) gene: a functional copy, cob 1, and a pseudogene, cob 2 (Kaleikau et al. 1992). In a survey of diverse rice lines, we found that cob 2 was absent in the wild abortive(WA)-type cytoplasmic male-sterile cytoplasm, but was present in the fertile lines. While cob 1 was conserved among all the lines, fertile and sterile, the cob 2 region was different in the fertile lines tested. The 5′ regions of most cob 2 loci were similar to cob 1 (about 4 kb of the flanking region and most of the coding region), but the 3′ region varied among different fertile lines. The point of divergence, the break-point, from the cob 1 sequence was conserved in all the cob 2 regions tested. In all the cob 2 regions, this break-point seems to be linked to the variable region of cob 2 through a conserved 192-bp segment, which is not a part of cob 1. It is proposed that the cob 2 regions could have been produced by recombination or insertion events involving cob 1 and the 192-bp segment which is present at different locations in the mitochondrial genomes of the various rice lines.     

Morphological responses by Bosmina longirostris and Eubosmina tubicen to changes in copepod predator populations during a whole-lake acidification experiment

Changes in zooplankton populations during the experimental acidilicationof Little Rock Lake provided an opportunity to examine specificmechanisms underlying the morphological responses of bosminidsto changing predation pressure. Two large copepods, Epischuralacustris and Mesocyclops edax, disappeared from the lake'sacidified basin in 1986 and 1989, respectively, while a smallercopepod predator, Tropocyclops extensus, increased during laterstages of acidification. The two bosminid species showed distinctlydifferent responses coinciding with the changes in copepod predation.Bosmina longirostris exhibited a significant decrease in mucrolength with the decline of M.edax and E.lacustris. Its meanbody and antennule length, however, did not change. We suggestthat the decoupling of B.longirostris mucro length and antennulelength may have been related to the persistence of the smallercopepod predator, T.extensus. Eubosmina tubicen showed no apparentresponse to declines in M.edax and E.lacustris abundance ineither mean mucro, antennule or body length. Allometric analysesindicated, however, that mucro length was related to size-dependentcopepod predation for both B.longirostris and E.tubicen.     

Effects of Prostaglandin E2 and Capsaicin on Behavior and Cerebrospinal Fluid Amino Acid Concentrations of Unanesthetized Rats: A Microdialysis Study

Abstract: Prostaglandin E₂ (PGE₂) delivered to the spinal cord produces an increased sensitivity to noxious (hyperalgesia) and innocuous (allodynia) stimuli. The mechanisms that underlie this effect remain unknown, but a PGE₂-evoked enhancement of spinal neurotransmitter release may be involved. To address this hypothesis, we examined the effect of PGE₂ on CSF concentrations of amino acids and also the modulatory effect of PGE₂ on capsaicin-evoked changes of spinal amino acid concentrations using a microdialysis probe placed in the lumbar subarachnoid space. Amino acids were quantified using HPLC with fluorescence detection. Addition of 1 mM, but not 10 or 100 µM, PGE₂ to the perfusate for a 10-min period (flow rate, 5 µl/min) evoked an immediate increase (80–100%) in glutamate (Glu), aspartate (Asp), taurine (Tau), glycine (Gly), and γ-aminobutyric acid (GABA) concentrations. Similarly, capsaicin infusion (0.1–10 µM) induced a dose-dependent increase in Glu, Asp, Tau, Gly, GABA, and ethanolamine levels. Significant increases in amino acid levels evoked by PGE₂ or capsaicin were associated with a touch-evoked allodynia. The combination of PGE₂ (10 µM) and capsaicin (0.1 or 1.0 µM) at concentrations that individually had no effect together evoked a significant increase (60–100%) in Glu, Asp, Tau, Gly, and GABA concentrations and produced tactile allodynia. These data demonstrate that spinally delivered PGE₂ or capsaicin substantially elevates CSF concentrations of both excitatory and inhibitory amino acids. The capacity of PGE₂ to enhance and prolong capsaicin-evoked amino acid concentrations may be one of the mechanisms by which spinal PGE₂ produces hyperalgesia and allodynia.     

Mutations of aspartate 103 in the Hm2 receptor and alterations in receptor binding properties of muscarinic agonists

Aspartate 103 (D103) in the third transmembrane domain of the Hm2 receptor was mutated to glutamate (D103E), asparagine (D103N), or alanine (D103A). As measured by [3H]-NMS, no significant binding was observed in D103A, while a 2-fold decrease in ligand affinity was seen in D103E and a 32-fold decrease in affinity was found in the D103N mutant. Examination of reference agonists showed greater loss of affinity in D103N than in D103E with the rank order of change being: L-607,207>carbachol>arecoline>pilocarpine>oxotremorine>McN-A-343. Of the novel 1-azabicyclo[2.2.1]-heptan-3-one oxime agonists examined, arylacetylene oximes showed little alteration in binding in either the D103E or D103N mutants, while the geometric isomers of several bicyclic aryl-ene-yne oximes showed significant changes in affinity, especially in the D103N mutant. Thus, overall size of the agonist and/or spatial orientation of the molecule within the binding pocket contribute to changes measured in binding.